EPA Report Collection
Regional Center for Environmental Information
U.S. EPA Region III
Philadelphia, PA 19103
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Ambient Toxicity and Chemical Characterization of Four Bayside creeks
of the Eastern Shore
2001
Chesapeake Bay Program
A Watershed Partnership
Chesapeake Bay Program
410 Severn Avenue, Suite 109
Annapolis, Maryland 21403
1-800-YOUR-BAY
http ://www.chesapeakebay.net
TJ.S. 1:1 PA Region III
I^iovun Center for Ermronmental
] G "vO Area Street (3PM52)
f hilu-ioJphia, PA 19103
Printed by the U.S. Environmental Protection Agency for the Chesapeake Bay Program
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Ambient Toxicity and Chemical Characterization
of Four Bay side creeks of the Eastern Shore
Morris H. Roberts, Jr.
Mark Luckenbach
Michael Unger
Virginia Institute of Marine Science
College of William and Mary
Gloucester Point, VA 23062
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ABSTRACT
The purpose of this study was to characterize selected bayside creeks of the Virginia portion of
the Delmarva Peninsula with respect to chemistry and toxicity. The peninsula is an area in which
the primary land use is agriculture. When compared to urbanized and industrialized areas, one
might expect fewer persistent impacts but with potentially severe intermittent impacts. To detect
persistent impacts, the ambient toxicity methods applied in the Chesapeake Bay area during the
past decade were used.
Severe intermittent impacts are generally undetected by these methods. Some agricultural
practices and rain events can produce severe pulses of toxicity. To detect intermittent or pulsed
impacts, in situ exposures with the grass shrimp, Palaemonetes pugio, were employed.
Four creeks were selected for examination. Three creeks (Hungar, The Gulf, and Old
Plantation), all in Northampton County, were known to have plasticulture of tomatoes and other
nightshade vegetables. The fourth creek (Onancock), in Accomac County, was selected as free
of plasticulture, but including an urbanized community (Onancock).
The chemical characterization of water from was limited to measuring cadmium, cobalt,
chromium, copper, mercury, nickel, lead, and zinc. At least two, and in two cases, five stations,
were sampled in each creek. In no case were any metals present in concentrations exceeding
water quality standards, acute or chronic. After observing water toxicity at all stations within
two creeks, stored samples were analyzed for chlorinated compounds and tributyltin. All
measurements were below the detection limits.
A more complete chemical characterization was made on sediment samples from two stations in
each creek. Among the metals analyzed (cadmium, cobalt, chromium, copper, manganese,
nickel, lead, and zinc), only nickel exceeded either of the benchmarks considered. Nickel
showed exceedances of the Effects Range Low in two of eight samples (Similar concentrations
were observed in three replicate samples from Carter's Creek, a reference site in the York River
drainage.). Similarly, no semi-volatile organic analytes or chlorinated organic analytes were
found to exceed sediment benchmarks, and most were non-detectable. Tributyltin, formerly used
in antifoulant paints for boats, both recreation and work, was also sought in the sediment without
a single instance of occurrence above the detection limit (1 ng/g). In contrast, TBT was detected
in sediment from the Poropotank River, a tributary of the York River where concentrations of 1.2
and 1.6 ng/g were observed.
The in situ test was conducted for one month at one site in each creek, a site selected to be at risk
from the primary land use for that creek. There was a single rain event that occurred in all four
creeks, a rain of 9.5 to 50 mm over two days. There was a slight increase in mortality associated
with this event in all creeks except Old Plantation. Other lesser rain events in three of the creeks
which occurred later in the month-long deployment were not associated with mortality.
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Water samples from Hungar Creek and The Gulf produced significant mortality in Palaemonetes
pugio at all stations, but in a subsequent test with Cyprinodon variegatus larvae, no mortality
was observed. Chemical analyses of the water in the first test did not reveal any material in
concentrations to explain the mortality.
Sediment samples from five sites in each creek were all found non-toxic using a Cyprinodon
variegatus embryo test or a Leptocheirusplumulosus amphipod test. This is consistent with the
lack of elevated concentrations of any analyte in sediment from these sites.
Mulinia lateralis larvae were exposed to water from the creeks and to pore water extracted from
sediment samples. The latter exposure has not to our knowledge been used previously. Only two
stations had water that was toxic, site 4 in Onancock Creek and The Gulf. No potential causative
agent was identified. Larvae exposed to pore water from seven sites, one or two in each creek,
had reduced survival and increased percentage abnormal. Again, there is no evidence to
implicate any particular causative agent.
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TABLE OF CONTENTS
ABSTRACT i
TABLE OF CONTENTS iii
INTRODUCTION 1
MATERIALS AND METHODS 3
Study Sites 3
Sampling of water and sediment 7
Analytical methods 8
Sediment characteristics 8
Grain Size 8
Metals 9
Water 9
Sediment 9
Organic Compounds 10
Water Samples 10
Sediment Samples 10
Tributyltin Analysis 11
Toxicity tests 11
In situ 11
Ambient Water tests 12
Palaemonetes larvae 12
Cyprinodon larvae 12
Mulinia larvae 13
Ambient Sediment tests 14
Cyprinodon embryos 14
Leptocheirus 10-day test 15
Mulinia embryo - pore water test 15
Reference Chemical Assays 16
RESULTS 17
Sediment characterization 17
Chemical characterization 17
Water 17
Sediment 23
Toxicological characterization 30
in situ test results 30
Toxicity of Water Samples 33
Toxicity of Sediment Samples 39
111
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Reference Chemical Tests 45
DISCUSSION 47
Acknowledgments 50
REFERENCES 52
APPENDIX A 55
APPENDIX B 69
IV
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INTRODUCTION
Tidal creeks of bayside Eastern Shore are areas where toxic substances are potentially introduced
only intermittently. A lack of data has not permitted a characterization of these areas with respect
to toxicity potential, and the absence of heavy industry makes this data limitation understandable.
However, there are potentially significant sources of contaminants in these tidal creeks, albeit
intermittent. In this predominantly agricultural area, major practices and crops include corn-soy
bean-wheat rotations, tomatoes and other vegetables under plasticulture, and conventional
vegetable culture, especially potatoes and melons. A diverse array of pest control chemicals are
used extensively in each case. Plasticulture in particular poses a significant risk because
rainwater drains off the crops, and accumulates exclusively between rows because of the plastic
mulch. Deliberate efforts are made to remove this excess water from fields. Finally various
management schemes are practiced to modulate the concentration of crop protectants before
water enters tidal creeks. The practices range from release over adjacent forested land with
ground infiltration to retention ditches and ponds capable of holding back substantial amounts of
water thereby providing time for the removal of toxic substances by a variety of processes.
In recent decades, a new aquaculrure industry has developed on the Eastern Shore to produce
hard clams. A number of hatcheries/nurseries have been established on various creeks, both
seaside and bayside. Juvenile clams produced in these facilities are then planted onto shallow
intertidal mud flats for grow-out. Proximity to the shorelines makes such grow-out areas
susceptible to adverse effects from land run-off, but perhaps more vulnerable are hatcheries and
nurseries which utilize water drawn from creeks as culture media. The early life stages in
hatcheries and nurseries (embryos, larvae, and early juveniles) are generally more sensitive to
adverse conditions and toxic materials than the late juvenile and mature clams found on intertidal
mud flats.
In hatcheries, minimal water treatment involves particle filtration and temperature modification.
By 1995, the industry had matured to the point that culture failure in hatcheries and nurseries
from poor techniques and limited expertise was reduced. Water quality is now considered the
principle cause of culture failure. Of possible water quality problems, toxic chemicals are of
major concern, though certainly not the sole issue. In 1996, allegations were made against a
major tomato producer using plasticulture as the cause of clam culture failures in a Gargathy
Creek hatchery on the seaside of the Eastern Shore and there were suspicions that similar events
might explain culture failures in other creeks, both seaside and bayside, notably in The Gulf.
Luckenbach et al. (1996) and Deitrich et al. (1996) provided some support for the allegations
regarding a possible role of runoff on clam hatchery/nursery failure in Gargathy Creek.
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Various biological assay methods using estuarine organisms are used to detect toxicity in
ambient water and sediment (Roberts & DeLisle, 1988; Hall et al., 1991, 1992, 1994;
Anonymous, 1997). Water and sediment samples collected at a point in time are evaluated for
toxicity using standardized methods. Test endpoints typically examined include both mortality
(or a surrogate) and growth or reproductive measures used in chronic tests. The latter endpoints
are used in order to enhance test sensitivity. Multiple species are tested as a hedge against
different sensitivities of various species to different chemicals.
These toxicity testing methods are accepted tools to take a snapshot of ambient toxicity but
assume that toxicity is uniform over time. These methods do not take into account intermittent or
pulsed inputs of toxic materials. Since water is sampled at a point in time, pulsed inputs can go
undetected if sufficient dilution occurs before sampling occurs. Therefore the methods are
inherently insensitive to short-lived adverse conditions.
A method using Palaemonetes adults exposed in situ was developed by Scott et al. (1987,1990)
for use in tidal creeks receiving agricultural run-off. In this procedure, the test animals are
continuously exposed to tidal creek water in cages and therefore subject to the effects of pulsed
releases of toxic materials. The endpoint in this test is mortality which limits test sensitivity, but
techniques to implement more sensitive endpoints such as growth or reproduction to tests
involving field exposure have not been developed for this species. This methodology was applied
in bayside creeks of Virginia in 1996 (Luckenbach et al., 1996), and proved valuable in detecting
ephemeral toxicity events related to rainfall events.
The objective of the present study was to characterize selected bayside creeks of the Eastern
Shore with respect to ambient toxicity, to perform a limited chemical characterization of water
and sediment, and to expand the chemical characterization of samples for which toxicity was
apparent. This approach to chemical characterization, while less comprehensive than the
characterizations typical of the decade-old ambient toxicity program of the Chesapeake Bay
program, was selected to avoid analyzing many samples with little likelihood of a significant
chemical load.
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MATERIALS AND METHODS
Study Sites
Four creeks were selected for study: Onancock, Hungar, The Gulf, and Old Plantation (Fig. 1).
These creeks are all less than 5 miles long and at most 0.6 miles wide. These creeks are all meso-
or polyhaline, and have little continuous freshwater input at the headwater. Therefore the creeks
are rather uniformly saline throughout their length. All four streams have sandy bottoms at the
mouth and for considerable distances upstream. These streams are shallow with depths generally
less than 6 ft except Onancock Creek which has depths reaching 9-10 ft in a few locations.
Onancock Creek, the most northern creek, is the longest of the four, totaling 4.6 miles in length
(Fig. 2). This is the only creek studied that lies in Accomac County. The city of Onancock is
located near the headwater at the confluence of two small streams draining agricultural lands to
the east. The city has a small sewage treatment plant discharging into the smaller of the two
streams about 0.2 miles upstream of the confluence. From, the confluence, the creek flows
generally westward to discharge into the Bay. The shoreline is fringed with agriculture (corn, soy
bean, wheat), woodland, private residences, and marsh land. This creek has the highest
concentration of residential commercial activity, but has no significant tomato culture.
Hungar Creek is the northernmost creek studied in Northampton county (Fig. 3). It is ca 4.6 miles
in length and has finer grained sediments over a larger area than any of the other creeks, although
sediments are still predominantly sandy. There is no significant community along its shoreline,
but there are residences in some areas. Land use is predominantly agricultural, with some small
amount of tomato culture.
The Gulf, near Eastville, VA, in Northampton County has no major community along its shores,
though the land extending south from the mouth along the Bay shore has a string of residences
(Fig. 4). This creek, the smallest of the four, is ca 1.9 miles long. There is at least one major
tomato farm along the southern bank. In this case the run off from the plasticulture fields is
collected in a retention pond that appears to be a dammed tributary of the creek. Water from the
pond is recirculated to the fields to provide irrigation. Whenever we have visited the pond, there
has been minimal discharge, though during heavy rainfalls, release of water to the creek is likely.
In some years, another tomato field is operated further upstream, but we lack any information
about how runoff from that field is managed. In addition there is other conventional agriculture
in this watershed, some woodland, as well as some residential development. Salt marshes are
limited within this watershed.
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Old Plantation Creek is the creek located farthest south in Northampton County (Fig. 5). It is
about 3 miles long, and follows a north-south axis inshore of the creek mouth oriented on an
east-west axis. The shoreline is variable, with wooded, marsh, open field and residential areas.
There did not appear to be any plasticulture activity in the watershed.
Five sampling sites were selected in each creek, located approximately equidistant from creek
mouth to creek head. These station locations are shown on Figures 1 to 4 along with important
landscape features. All stations were quite shallow, with a maximum depth of ca 3 ft in most
creeks and 9-10 ft in Onancock Creek. In addition, a site is indicated in each creek at which the
in situ test was performed. These latter sites were selected deliberately to be near locations where
pulsed inputs of contaminants were likely in the event of a significant rain event (>%") and with
easy access by road.
38°00'
37°30'
llie Gulf
Creek
Atlantic Ocean
Old Plantation
Creek
Figure 1
Map of Eastern Shore, Virginia showing some
principal geopolitical features and locating the four
creeks sampled in the present study.
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r
Figure 2 Onancock Creek with all stations located by solid circles. The site of the in situ
test, located on the North Branch, is indicated by an 'x'
1000 2000 Meters
Figure 3 Hungar Creek with all stations located by solid circles. The
site of the in situ test is located by an 'x'.
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Figure 4 The Gulf with all stations indicated by solid circles. The site of the in situ study is
coincident with Station 2, and is immediately downstream of the discharge from a
retention pond serving two large tomato fields in which plasticulture is practiced.
Figure 5 Old Plantation Creek with all
stations located by solid circles.
Station 2 was relocated by error
to location 2' indicated by the
open circle. The site of the in
situ test iseindicated by an 'x'.
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Sampling of water and sediment
The eastern shore has been predominantly agricultural during this century coupled with boating
activity in association with the various creeks fringing the peninsula. Therefore it was decided to
make as comprehensive a toxicological characterization as possible and to limit the chemical
characterization to a subset of stations and to those chemicals likely to be found in the matrix
being examined as a result of known anthropogenic activities on the Eastern Shore. Samples
were collected at all stations for the full suite of chemical analyses (exclusive of the sampling for
Mulinia lateralis embryo tests), but only those from stations 2 and 4 in each creek were analyzed
as part of the characterization effort.
Water samples were collected at a depth of 18 inches from the surface, which at many stations
represented mid-depth. An 18 liter sample was collected for toxicity tests. At the same time, a 4
liter sample was collected for analysis of organic analytes and placed in a chemically clean amber
glass bottle, chilled on ice during transport, and stored at 4°C on arrival at the Gloucester Point
laboratory before extraction. Additionally, 1 liter samples were collected into high density
polyethylene bottles for metals analysis. These samples were also placed on ice during transport,
filtered and acidified for metals analysis. The filtered, acidified samples were stored at 4 °C
pending analysis. Water samples were used in toxicity tests within 48 hr of collection.
Sediment samples were collected at each station using a stainless steel petit Ponar grab, with only
the surface 1-2 cm of sediment retained for analysis. At each site, multiple grabs were taken to
provide sufficient sediment for analysis of organic chemicals, metals, grain size, total organic
carbon (TOC), pore water ammonia, AVS and toxicity to the selected test species. Sediments for
organic analyses were placed in glass mason jars, kept on ice during transport, and frozen on
arrival at the laboratory pending extraction. The bulk of the sediment was returned to the
laboratory in 18 liter plastic pails with lids. On arrival at the laboratory, the samples were placed
in a 4°C cold room. Subsampling for various analyses and processing for toxicity tests was
initiated within 24 hours of arrival at the laboratory. Sediment was prepared for toxicity tests
with amphipods by press sieving through a 500 |im mesh stainless steel screen to remove any
resident amphipods or invertebrates that might prey on the amphipods. Additional sediment was
prepared for tests with fish embryos by sieving through a 2.0 mm mesh stainless steel screen to
remove large animals that might prey on fish embryos. The prepared sediments were returned to
the cold room pending completion of preparations and initiation of each test. All tests with
sediment were initiated within 7 days of collection.
Unscreened sediment samples were submitted to the VIMS Analytical Services Center for grain
size analysis. Samples were also submitted to the Analytical Services Center for TOC analysis
along with pore water samples for ammonia analysis. Two additional subsamples from each site
were placed in whirl pac containers and chilled prior to analysis one for sediment metals and one
for acid volatile sulfides (AVS).
Samples for ambient water toxicity tests were collected from Hungar Creek and The Gulf on 21
September 1998 and from Onancock and Old Plantation Creeks on 23 September 1998. These
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samples were tested with Palaemonetes pugio larvae. Sampling was staggered primarily to
accommodate the time needed to establish the toxicity tests with limited personnel. The full suite
of tests with this species was accomplished on this schedule because sufficient larvae were
produced daily for the tests in this time window and availability of ovigerous females later in the
year was unlikely. The stations were resampled on 12 October 1998 and 14 October 1998 to
provide ambient water for the tests with Cyprinodon variegatus larvae.
Samples for sediment toxicity tests were collected from The Gulf on 30 October 1998 for an
initial test with both C. variegatus embryos and the amphipod Leptocheirusplumulosus. While
we had considerable experience with the latter species, the test with fish embryos had not been
performed previously. This experiment yielded inadequate data because of larval escape from the
egg chambers (data are included in the appendix but not analyzed), and sampling of The Gulf
was rescheduled. The egg baskets were refabricated with 400 |J,m mesh screen before additional
tests were done.
Sediment samples were collected from Old Plantation Creek on 17 November 1998 and the
sediment tests with both C. variegatus and L. plumulosus were performed. In this case the tests
were successful with no larval escapement and with acceptable control survival. The sampling
and testing schedule was interrupted by the holiday season and resumed in February 1999 with
sediment collections from The Gulf and Onancock Creeks on 8-9 February 1999, and from
Hungar Creek on 16 March 1999.
A major limitation of the sampling and toxicity testing scheme was the non-synoptic sampling
necessitated by logistics. For the Mulinia lateralis tests, a synoptic sample set of water and
sediment was accomplished on 26 April 1999, allowing both water and pore water tests to occur
simultaneously with a single spawn of Mulinia involving 3-4 females and multiple males.
Analytical methods
Sediment characteristics
Grain Size
A weighed aliquot of sediment from each site was analyzed for grain size and percent total
organic carbon (TOC) using standard gravimetric methods. All these analyses were performed by
the VIMS Analytical Services Center. Sufficient sediment (typically 200 g) was centrifuged at
5000 rpm (7000 g) and the pore water decanted. The pore water sample was then analyzed for
ammonia by the VIMS Analytical Services Center.
Acid volatile sulfide (AVS) in samples of sediment from each collection site was measured using
the diffusion method described by Leonard, Cotter and Ankley (1996) who developed it as an
efficient alternative to the purge-and-trap method of Di Toro et al. (1990, 1992). This diffusion
method was based on earlier work of Brower and Murphy (1994) and Hsieh and Yang (1989).
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Though it is common to measure the simultaneously extracted metals (SEM), we did not elect to
do so. Sulfide was measured with a sulfide specific electrode (Orion Model 9616) connected to
an Orion Expandable Ion Analyzer EA920. The electrode was calibrated daily using fresh sulfide
stock solutions.
Metals
Water
Water samples were filtered through a 0.47 urn glass fiber filter and the filtrate containing
dissolved metals was acidified (pH<2) and stored at 4°C pending analysis. A cobalt-
coprecipitation method modified from Boyle and Edmond (1975) was used to extract metals
from water samples, thereby reducing the background noise resulting from other salts in the
samples and facilitating a twenty-fold concentration of analyte. A 100 g aliquot of each acidified
sample water was amended with acetate buffer. Each sample received 4 ml of a chelator, (APDC
or 1 pyrrolidiine carbodithioic acid ammonium salt) followed by solution 2 ml of CoCl2 prepared
according to Kraus & Moore(1953). After standing for a minimum of 30 minutes to allow the co-
precipitate to form and scavenge other dissolved metals present in the sample, the precipitate
containing the analyte metals samples is collected on a 0.47 \im polycarbonate filter. The filter
was placed in precleaned LPE screw cap vials, acidified with 3N HC1 and sonicated for 30
minutes in a warm water bath to dissolve the precipitate. Once dissolved, the resultant solution
was diluted with deionized water and stored at room temperature until analyzed by atomic
absorption spectrophotometry. For The Gulf and Hungar Creek samples, in which case water
from all 5 stations was analyzed, three additional aliquots of water from Stations 1 and 5 were
used for standard additions. These aliquots received 100, 200 or 500 \il spikes of a standard
metals mixture containing cadmium, copper, nickel, zinc, chromium, lead, and iron. These
spiked samples were then analyzed in the same manner as the unspiked samples. Water from two
sites was used for spiked additions to assess whether a reliable curve could be produced using a
single sample from a creek. The samples used for the spiked additions curves were selected to
reflect our suspicion that upstream samples might differ those from the creek mouth stations. For
Onancock and Old Plantation Creek samples, in which case only samples from stations 2 and 4
were analyzed, aliquots of all samples received spikes.
Mercury was determined by FIA cold vapor atomic absorption on aliquots of the original filtered
samples. Zinc and cobalt were determined by direct flame atomic absorption. All remaining
metals (cadmium, copper, chromium, nickel, and lead) were measured by flameless atomic
absorption using the extracted metal sample. A Pd/Mg universal matrix modifier was used for
graphite furnace analyses.
Sediment
Freeze dried sediments were microwave digested with concentrated nitric acid. The resulting
digest was then analyzed by flame atomic absorption spectrometry or cold vapor atomic
absorption spectrometry for mercury.
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Organic Compounds
Ambient toxicity may not be related solely to "priority pollutants" as defined by EPA
(Allred and Giesy, 1985). We therefore analyzed for a broader array of potentially toxic chemical
agents. "Standard" protocols often overlook some organic compounds and their degradation
products which in themselves may be more toxic than precursors. Samples were analyzed for
select priority pollutants and specific pesticides analyzed in previous AMBTOX studies but we
also used a semi-quantitative relative retention indices analytical approach to search for a broader
array of chemicals (Greaves, et. al, 1991). Using this technique, it is possible to assign peak
retention identities to unknown compounds that may be then identified by mass spectrometry
when concentrations are sufficient. A listing of searchable compounds in the Aromatic Retention
Indices (ARI), Halogenated Retention Indices (HRI), and Polar Retention Indices (POI) are
presented in Appendix A, Tables A1-A9 . Performance of these methods was evaluated through
the analysis of Standard Reference Materials (SRM) and matrix spikes.
Water Samples
Water samples were acidified with HC1, spiked with surrogate standards, and extracted
three times with 100 ml of dichloromethane. The extract volume was reduced, the solvent
exchanged to hexane and internal standards added. Analysis of extracted samples was performed
on a Varian gas chromatograph equipped with an electrolytic conductivity detector (GC-ELCD).
The instrument calibration was confirmed prior to sample analysis using four point calibration
standards representing typical PCB and chlorinated pesticide analytes. The resulting
chromatograms were analyzed using a halogen retention index (HRI) to identify tentatively any
peaks found in the samples. Mass spectrometric analysis was conducted on representative
samples. Chromatograms were searched to confirm the presence of the predetermined set of
priority pollutants using retention time and El spectra. Spectra were compared with computer
searchable spectra published in the NIST Standard Reference Database, in-house library spectra
generated from standards and spectra published in articles or books. Blank and fortified samples
were analyzed in addition to environmental samples.
Sediment Samples
Sediment samples were analyzed by the VIMS protocol for toxic organic chemicals which
is described in detail in the SOP (Greaves, et. al, 1991). Briefly, sediments were freeze-dried,
spiked with surrogate standards, and extracted with dichloromethane with an Accelerated Solvent
Extractor (ASE). The resulting extracts were fractionated by GPC and silica gel, spiked with
internal standards and analyzed for aromatic or heterocyclic compounds by capillary gas
chromatography with flame ionization detection (GC/FID) or gas chromatography mass
spectrometry (GC/MS) in the full scan electron ionization mode. Chlorinated hydrocarbons were
analyzed by capillary gas chromatography with electrolytic conductivity detection (GC/ELCD).
Blank samples, fortified samples and SRM 194la were analyzed concurrently with
environmental samples.
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Tributyltin Analysis
Water and sediment samples were processed by an adaptation of the methodology
published previously for butyltins in water samples, sediments and tissues (Unger et al, 1986,
Unger, 1996 ). Samples were extracted with hexane/tropolone and derivatized with excess
Grignard reagent (n-hexyl magnesium bromide). Remaining Grignard reagent was neutralized
with 6 N HCL and the hexane layer removed and cleaned up by open column chromatography
with Florisil®. Extracts were reduced in volume under dry nitrogen, spiked with tetrabutyltin
internal standard and analyzed by gas chromatography with flame photometric detection.
Toxicity tests
In situ
In situ tests were accomplished between 5 August 1998 and 23 August 1998 using a modification
of the method described in Luckenbach et al. (1996). At one site in each creek, three cages were
deployed on 5 August, each cage containing 12 individually housed shrimp, for a total of 36
animals placed at each study site. Individual caging is necessary to prevent cannibalism that
would otherwise be likely whenever a molting event occurred. The individual compartments
consisted of mesh-covered tackle boxes (3 mm mesh) that allowed a relatively free exchange of
water between the chambers and the water ambient at the station. The trays were deployed by
suspending them at the surface from a PVC floatation ring. Every other day, the cages were
examined for survival of shrimp, and the mesh cleaned by gentle brushing if necessary. On each
observation day, air and water temperature, salinity and dissolved oxygen concentration were
determined. pH was measured with a hand-held meter during the middle portion of the study
period. The time of observation, tidal stage, wind direction and speed and percent cloud cover
were also recorded. The first in situ exposure was terminated on August 13, and a new set of
animals was deployed using the same procedures on 15 August, remaining in place until 23
August.
Dead shrimp decomposed rapidly under the prevailing conditions. A shrimp was recorded as
dead if a dead body (whole or part) was found. If no body was found, the shrimp was classed as
missing. A shrimp could become missing through escape (during observation), movement to an
adjacent cell in the exposure tray, or death and decomposition between observation times. In the
case of movement to an adjacent cell, the shrimp were left in the adjacent cell until the end of the
exposure period rather than returned to the original cell. Only 16 animals were recorded as
missing out of 288 animals deployed (4 stations, 36 per deployment, 2 deployments) (5.6%). Of
these, 12 appeared in other chambers (4.2%) with only 4 either unobserved dead or escapees
(1.4%).
1!
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Ambient Water tests
Palaemonetes larvae
Larvae of Palaemonetes were obtained from ovigerous female shrimp collected from Sarah
Creek (at Sarah's End Lane off Route 216) and held in the laboratory in large tanks until needed.
Seven to 8 days before a scheduled test, approximately 100 ovigers were selected, judged to be
near hatch based on color of the egg mass and placed in hatching baskets in 10 gal aquaria of 15
psu filtered York River water. Sixteen hours later, the resultant larvae were harvested and placed
in 4 liter glass jars in 15 psu water for culture. Each day, newly hatched Anemia nauplii were
added to the culture jars as food after siphoning out any residual dead nauplii and debris from the
jars. Larvae were used for tests after 7-8 days in culture. When the cultures were approximately 5
days old, the daily water change was done using 15 psu Hawaiian Marine Mix to acclimate the
zoeae to the control water used in the test.
Water for a test was processed to adjust salinity and temperature beginning the day before the test
(one day after collection). On the initial day of the test, water was placed in glass jars with 4
laboratory replicates of each treatment. Negative control samples consisted of artificial seawater
at 15 psu prepared by dissolving Hawaiian Marine Mix in deionized water. Positive controls
were cadmium chloride solutions prepared at the previously measured 96 hr LC50.
All jars were placed in a 25°C water bath over night preparatory to introduction of shrimp larvae.
Larvae were distributed sequentially into small weigh pans, one larva at a time, until all pans
contained 10 larvae. The contents of the pans were then placed into randomly chosen test
containers, and the containers placed in the water bath according to a stratified random scheme,
one replicate of each treatment per stratum.
On day 0 and day 8 of the test, the temperature, salinity, dissolved oxygen concentration, and pH
were measured in water from one replicate of each treatment. On all other days, the temperature
in a different replicate of each treatment was measured and recorded along with the minimum
and maximum temperature of the water bath. Each day, the number of live larvae in all replicates
and treatments was counted and any dead larvae removed. At the same time, a 50% water change
was accomplished using water prepared the day previous and allowed to temperature equilibrate
over night.
At the end of the exposure period, larvae were counted, rinsed with deionized water, and placed
in tared aluminum weigh pans, one pan per treatment replicate. The larvae were dried at 103°C
for 72 hours, then cooled in a desiccator, and weighed to the nearest 0.1 mg.
Cyprinodon larvae
Newly hatched fish larvae were purchased from Aquatic Biosystems (3800 Weicker Dr., Fort
Collins, CO 80524) for delivery immediately before a test. On arrival in the laboratory, the fish
were placed in large plastic pans of the shipping water and the salinity and temperature gradually
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adjusted to 15 psu and 25°C respectively over a 24 hour period. Any dead or damaged larvae
were removed.
Water was removed from the cold room a day prior to start of the test, the salinity adjusted to 15
psu, and distributed among the test beakers. Four laboratory replicates were established for each
sample site, negative controls and positive controls. Negative controls consisted of artificial
estuarine water prepared from Hawaiian Marine Mix and deionized tap water. Positive controls
consisted of a cadmium chloride solution prepared at a concentration intended to be a previously
determined LC50.
All beakers were placed in a 25° C water bath over night preparatory to introduction offish
larvae. Larvae were distributed sequentially into small weigh pans, one larva at a time, until all
pans contained 10 larvae. The contents of the pans were then placed into randomly chosen test
containers, and the containers placed in the water bath according to a stratified random scheme.
On day 0 and day 8 of the test, the temperature, salinity, dissolved oxygen concentration, and pH
were measured in water from one replicate of each treatment. On all other days, the temperature
was measured and recorded along with the minimum and maximum temperature of the water
bath. Each day, the number of live larvae was counted and any dead larvae removed. At the same
time, a 50% water change was accomplished using water prepared the day previous and allowed
to temperature equilibrate over night.
At the end of the exposure period, larvae were counted, rinsed with deionized water, and placed
in tared aluminum weigh pans, one pan per treatment replicate. The larvae were dried at 103°C
for 72 hours, then cooled in a desiccator, and weighed to the nearest 0.1 mg.
Mulinia larvae
Adult Mulinia lateralis were procured from the Marine Biological Laboratory (Aquatic
Resources Division, Woods Hole, MA 02543) and maintained in 10 gal aquaria with a 1-2 cm
layer of sand on the bottom and about 8 gal estuarine water at 25 psu. Each day, 1.5 liters of
resuspended Isochrysis galbana was added as food for the clams. Clams buried in the sediment
and actively pumped to clear the algae in 3-4 hours. Clams that failed to burrow were removed
and discarded.
When all water (and pore water) samples were ready for toxicity analysis, clams were removed to
8 inch glass fingerbowls and placed in a heated water bath to raise the temperature to ca 28 °C.
The temperature in the bowl was cycled up to 28°C, down to 12 °C and back to 28°C until one or
more males released sperm. The males were isolated in separate bowls and allowed to continue
sperm release. Small aliquots of sperm suspension were pipetted into the incurrent flow of
remaining clams to facilitate female spawning. As females were identified, they were isolated in
separate bowls to collect the eggs. When it was judged that sufficient eggs were available, eggs
from several females were composited and a composite sperm sample added to accomplish
fertilization. Once fertilized eggs were obtained, the eggs were washed on a 35 urn nylon screen
13
-------
to remove excess sperm and resuspended and aliquots were counted. From the counts, the
volume of egg suspension necessary for each test replicate was calculated.
Water (and pore water) samples (10 ml) tempered to 25 °C were distributed to each of three 20
ml glass scintillation vials (laboratory replicates), and the appropriate volume of egg suspension
added to each to yield an egg density of 30 eggs/ml. In addition to field samples, negative control
samples were tested (along with pore water obtained from reference site sediment collected from
Carter's Creek and Poropotank River). Simultaneously, a cadmium chloride toxicity test was
conducted using the same volume of test solution and embryos from the same spawn. All vials
were incubated for 48 hours at 25°C. At that time, each vial was fixed with buffered formalin,
and embryos were examined over several weeks to determine number of survivors and percent of
survivors achieving the normal straight-hinge stage.
Ambient Sediment tests
Cyprinodon embryos
Twenty-four hour old embryos were obtained from Aquatic Biosystems. These embryos were
produced by spawning of fish stimulated with human gonadotrophic hormone. On receipt, fertile
eggs were isolated from the embryos received and held overnight preparatory for a test. As will
be seen in the results section, the percent hatch of these selected eggs in the control treatments
was often in the 60-70% range and therefore did not meet the planned acceptability criterion for
control hatch. However, the tests could not be repeated within the budget and time constraints.
The results were consistent across laboratory replicates, an indication that the problem was a
function of marginal egg condition, not technique. In no case did we observe overgrowth with
fungi or other indications of a flawed exposure. In two instances, eggs not used in the test were
maintained to measure % hatch when not exposed; in these cases, the % hatch was comparable to
that for control sediment exposures.
For exposure tests, four laboratory replicates of each sediment treatment were prepared in 1 liter
glass beakers and overlain with 1500 ml of artificial sea water at 15 psu salinity and 25 °C. For
each treatment replicate, embryos were placed in an egg basket with the bottom screen resting
closely on the sediment to insure egg contact with sediment while not allowing burial. The egg
baskets were constructed from a 4" PVC pipe section about 8" long. Two windows were cut in
the sides of each basket and covered with nylon mesh. A piece of nylon mesh was held across the
open end of the basket with a nylon pipe coupler. The initial set of baskets was prepared with
mesh screen which proved too large to retain newly hatched larvae resulting in failure of the
initial fish embryo test. Therefore, egg baskets were reconstructed with 400 |im mesh nylon.
On day 0 and day 8 of the test, the temperature, salinity, dissolved oxygen concentration, and pH
were measured in water from one replicate of each treatment. On all other days, the temperature
was measured and recorded along with the minimum and maximum temperature of the water
bath. Throughout the tests, a slow stream of air bubbles was introduced into each vessel from a
high-volume-low pressure laboratory air system to insure maintenance of dissolved oxygen levels
14
-------
and to minimize accumulation of ammonia. Each day, after the egg baskets were removed to
determine hatch and survival, a 50% water change was accomplished using water prepared the
day previous and allowed to temperature equilibrate over night.
Daily during the test, the egg baskets were removed from each treatment, eggs (and hatched
larvae) were removed with large-bore pipet, placed in a fingerbowl, and examined
microscopically on a light table. Obviously non-developing embryos showing significant decay
were removed. The number hatched on each day was recorded along with the survival of
hatchlings. Some tests were terminated before the full 10-day exposure period based on a
criterion that all viable eggs in the negative control replicates had hatched two days previously.
Endpoints measured were percent hatch and post-hatch larval survival.
Leptocheirus 10-day test
Amphipods for the tests were produced in cultures maintained in the toxicology laboratory at
VIMS. Twenty-four hours before a test, amphipods of the appropriate size were isolated from
cultures by gradient screening, excluding animals collected on a 1.0 mm screen and those passing
through a 0.5 mm screen. The procedure produces a more uniform sized test population than
other approaches used to produce 7-day old animals.
The day before establishing a test, a 2-cm deep layer of sediment was placed into each container
and overlain with 500 ml of 1 |im filtered York River estuarine water (adjusted to 15 psu with
distilled water). Five laboratory replicates were prepared of each sediment sample. In addition to
the field samples from the eastern shore creeks, sediments from two reference sites, one in the
Poropotank River (corresponding to the control site used by AMRL) and one in the mouth of
Carter's Creek (both tributaries of the York River) were prepared as reference samples.
Reference sediments were subsampled for various analyses in the same manner and time as
experimental site samples, but were not collected within the same time window as the
experimental samples.
On day 0 and day 10 of the test, the temperature, salinity, dissolved oxygen concentration, and
pH were measured in water from one replicate of each treatment. On all other days, the
temperature was measured and recorded along with the minimum and maximum temperature of
the water bath. Throughout the tests, a slow stream of air bubbles was introduced into each vessel
from a high-volume-low pressure laboratory air system to insure maintenance of dissolved
oxygen levels and to minimize accumulation of ammonia.
At the end of the exposure period, the amphipods were screened from the sediment, counted,
rinsed in deionized water, and placed into tared aluminum weigh pans. The amphipods were
dried at 103°C for 72 hours, then cooled in a desiccator, and weighed to the nearest 0.1 mg.
Mulinia embryo - pore water test
The pore water tests were performed simultaneously with the ambient water tests for this species
15
-------
using the procedures described previously.
Reference Chemical Assays
For Cyprinodon variegatus larvae, Palaemonetes pugio zoeae, and Leptocheirus plumulosus,
standard toxicity tests were performed with cadmium chloride as the reference toxicant, chosen
for consistency with historical methods used in the ambient toxicity testing program. The basic
method was that described in ASTM Designation E729. All treatments were tested in duplicate.
The data for C. variegatus larvae were assumed to represent the status of the embryos in the
absence of a widely accepted standard test applicable to the eggs. For the M. later alls, the test
method was that described in ASTM Designation E724, using the minimal test solution volume
recommended, i.e. 10 ml in 20 ml scintillation vials. All treatments were tested in triplicate.
16
-------
RESULTS
Sediment characterization
The Gulf, Hungar, and Old Plantation creeks are characterized by sandy substrates downstream,
with varying amounts of silt/clay admixture at the upstream stations (Table 1). In contrast,
Onancock Creek sediments were sandy (>93% sand) both downstream (stations 1 and 2) and
upstream (stations 4 and 5), but silty in the middle (station 3: 8.6 % sand, 64% silt). Predictably,
total organic carbon (TOC) at the sandy stations was low (<0.5 %), but variable at non-sandy
stations; at the stations dominated by silt/clay (>90% silt/clay), the TOC was high (1.9-6.4%).
Stations with intermediate grain size distributions have intermediate TOC concentrations. Acid-
volatile sulfide concentrations showed no clear relationship to grain size distributions nor to
TOC. Pore water ammonia concentrations were also highly variable, but were consistently above
5 mg/1 in silty sediments, and ranged from <1 to 9 mg/1 in sandy sediments.
Chemical characterization
Water
Ammonia and nitrite concentrations measured on ambient water samples collected during the
sediment sampling events were low, between 0.010 and 0.026 mg/1 for ammonia and 0.001 and
0.006 mg/1 for nitrite (Table 2).
Water samples were analyzed for cadmium, chromium, copper, nickel, mercury, lead and zinc.
The measurements are arrayed in Table 3 along with values for field or travel blanks and distilled
water blanks. The method detection limit and limit of quantitation are included for each metal
along with the US EPA chronic Water Quality Criterion (EPA, 1987) and the acute and chronic
Virginia Water Quality Standards.
17
-------
Table 1. Sediment characteristics at each station. Sediment from The Gulf (10/30/98) used for
failed toxicity test, and included for comparative purposes only.
Station
Onancock 1
Onancock 2
Onancock 3
Onancock 4
Onancock 5
Hungar 1
Hungar 2
Hungar 3
Hungar 4
Hungar 5
The Gulf 1
(10/30/98)
The Gulf 2
The Gulf 3
The Gulf 4
The Gulf 5
The Gulf 1
(2/8/99)
The Gulf 2
The Gulf 3
The Gulf 4
The Gulf 5
Old Plantation 1
Old Plantation 2
Old Plantation 3
Old Plantation 4
Old Plantation 5
Percent
TOC
0.07
0.04
3.20
0.26
0.49
0.08
2.12
1.92
0.45
4.34
0.04
0.06
0.77
2.79
3.81
0.04
0.02
0.14
4.21
6.38
0.02
0.05
0.71
0.66
1.28
NH4
(mg/kg)
0.978
1.409
8.143
3.163
4.885
6.00
3.01
4.26
6.56
7.98
2.24
1.62
6.43
6.13
5.17
0.406
0.981
5.982
5.573
4.149
Acid
Volatile
Sulfide
0.00
0.00
1.69
2.30
8.20
0.00
6.05
8.20
1.08
2.80
0.00
0.00
1.35
2.75
1.16
0.02
0.00
4.35
4.80
10.45
0.00
0.18
1.56
0.51
0.30
Percent
Moisture
17.0
62.8
55.8
24.7
84.4
16.6
16.9
33.8
61.7
71.9
20.9
19.7
36.8
30.9
42.9
Percent
Gravel
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
1.35
0.00
0.00
0.60
0.00
0.00
0.00
0.00
0.05
0.43
0.00
0.00
0.36
0.00
Percent
Sand
93.94
95.89
8.62
93.66
96.01
97.80
14.10
23.10
86.30
2.30
99.00
97.10
81.90
2.91
13.09
97.59
98.16
88.73
2.86
7.26
98.66
98.55
60.69
80.26
54.48
Percent
Silt
3.12
2.15
64.33
4.05
2.83
0.80
41.40
37.90
5.50
40.70
0.22
0.34
9.45
48.32
49.20
1.24
0.92
7.54
65.47
37.77
0.26
0.48
26.06
10.59
26.85
Percent
Clay
2.93
1.96
27.05
2.29
1.16
1.40
44.50
39.00
8.20
57.00
0.74
1.22
8.63
48.76
37.12
1.17
0.92
3.73
31.67
54.92
0.66
0.96
13.25
8.78
18.68
18
-------
Table 2. Ammonia and nitrite concentrations in ambient water, selected sampling dates.
Onancock 1
Onancock 2
Onancock 3
Onancock 4
Onancock 5
Hungar 1
Hungar 2
Hungar 3
Hungar 4
Hungar 5
The Gulf 1(10/98)
The Gulf 2
The Gulf 3
The Gulf 4
The Gulf 5
The Gulf 1 (2/99)
The Gulf 2
The Gulf 3
The Gulf 4
The Gulf 5
Old Plantation 1
Old Plantation 2
Old Plantation 3
Old Plantation 4
Old Plantation 5
mg NH4/1
0.010
0.014
0.018
0.022
0.022
0.011
0.011
0.012
0.016
0.025
0.026
0.023
0.014
0.011
0.014
0.015
0.015
0.014
0.020
0.014
0.013
0.012
0.010
0.012
0.054
mg NO2/1
0.002
0.002
0.004
0.004
0.005
0.001
0.002
0.002
0.003
0.003
0.001
0.001
0.001
0.002
0.003
0.001
0.001
0.002
0.003
0.005
0.001
0.001
0.002
0.003
0.006
19
-------
Table 3. Dissolved metal concentrations observed in water samples (in ng/1). Values are uncorrected for blank
concentrations. All field blank concentrations as well as a distilled water blank are included in the table.
Sample
Onancock 2
Onancock 4
Onancock Field Blank
Hungars 1
Hungars 2
Hungars 3
Hungars 4
Hungars 5
Hungars Field Blank
The Gulf 1
The Gulf 2
The Gulf 3
The Gulf 4
The Gulf 5
The Gulf Field Blank
Old Plantation 2
Old Plantation 4
Old Plantation Field Blank
Distilled Water Blank
Method Detection Limit
Limit of Quantitation
US EPA Water Quality Criterion,
Chronic (US EPA, 1987)
Virginia Water Quality Standard, Acute
(VAC 25-260- 140)
Chronic
Cd
0.14
0.11
<0.11
0.12
0.12
0.11
0.11
<0.11
0.11
0.22
0.27
0.17
0.45
0.24
0.13
0.19
0.11
0.17
0.13
0.11
0.38
9.3
43
9.3
Co
<0.99
1.72
<0.99
<0.99
<0.99
1.95
O.99
<0.99
<0.99
<0.99
<0.99
<0.99
<0.99
<0.99
<0.99
1.27
1.00
<0.99
<0.99
0.99
3.33
-
-
-
Cr
O.35
1.22
0.65
0.58
0.66
0.53
0.63
<0.35
0.68
0.57
0.75
0.87
0.83
O.35
0.44
~
1.38
0.67
1.06
0.35
1.16
50
1100
50
Cu
0.39
1.01
<0.10
0.49
0.45
0.42
0.45
0.42
<0.10
0.52
0.75
0.54
1.68
0.21
<0.10
0.52
0.56
<0.10
<0.10
0.10
0.35
2.9
5.9
3.8
Ni
0.98
0.98
<0.56
1.08
1.10
1.11
1.11
1.33
<0.56
0.95
0.93
1.33
1.12
1.57
<0.56
0.83
0.94
<0.56
<0.56
0.56
1.86
8.3
75
8.3
Hg
<0.50
<0.50
<0.50
<0.50
<0.50
O.50
<0.50
O.50
<0.50
<0.50
<0.50
<0.50
<0.50
<0.50
<0.50
<0.50
<0.50
<0.50
0.76
0.50
0.79
0.025
2.1
0.025
Pb
<0.21
0.29
<0.21
0.28
<0.21
<0.21
<0.21
<0.21
<0.21
~
<0.21
<0.21
<0.21
<0.21
<0.21
0.60
0.26
O.21
<0.21
0.21
0.71
5.6
240
9.3
Zn
<20.2
<20.2
<20.2
<20.2
<20.2
<20.2
<20.2
<20.2
<20.2
<20.2
<20.2
<20.2
<20.2
<20.2
<20.2
<20.2
<20.2
<20.2
<20.2
20.15
67.15
86
95
86
"-" means that there is no Water Quality Criterion or Water Quality Standard for comparison.
20
-------
The field blanks exhibit substantial unexplained variability. For these reasons, no corrections
have been made on the field measurements. Clearly, however, the measured concentrations of
most metals were equal to or greater than the field blanks. If corrected for the field blanks, the
concentrations would generally be below the method detection limit. Even if one assumes that
the field blanks were compromised (in which case the sample concentrations are the maximum
possible), uncorrected metal concentrations were never above the EPA chronic WQC for a saline
water or the Virginia chronic WQS.
Water samples indicating toxicity were extracted and analyzed for chlorinated
compounds. Overall, concentrations were very low with no compounds above detection limits
(Tables 4). All sample chromatograms were similar to each other in pattern composition and do
not contain elevated concentrations of known contaminants. Tabular results from fortified sample
analysis (Appendix A) show recoveries for a wide spectrum of contaminants. Deionized water
blanks, sample replicates, and a field blank, all had no quantifiable analytes. Mass spectrometric
analysis was conducted on representative samples. Complete printouts of the HRI for these
samples can be found in Appendix B.
Aliquots of water from 2 sites in all four creeks were analyzed for the organometallic
antifouling agent, tributyltin (TBT) and it's degradation products, dibutyltin and monobutlytin.
These are all sufficiently water soluble that if present in toxicologically significant amounts, they
would be detectable. None of the analyzed samples contained butyltins above the detection limit
of 1 ng/I and matrix spiked samples (5 ng/L) showed 100% recovery of TBT (Table 5).
21
-------
O
(0
S
£
I/I
t/)
28s
3 o
£
£
-------
Table 5 Butyltin concentrations in water from all sites in two creeks, Hungar and The Gulf,
exhibiting toxicity to shrimp larvae..
Water Samples
Detection Limit 1 ng/L
All concentrations as Cation in ng/L
Matrix Spike 5 ng/L
Sample
Hungar Creek Site 1
Hungar Creek Site 2
Hungar Creek Site 3
Hungar Creek Site 4
Hungar Creek Site 5
The Gulf Site 1
The Gulf Site 2
The Gulf Site 3
The Gulf Site 4
The Gulf Site 4 Replicate
The Gulf Site 5
Matrix Spike
Matrix Spike Duplicate
Field Blank
Lab Blank
Lab Blank
Date Collected
9/21/98
9/21/98
9/21/98
9/21/98
9/21/98
9/21/98
9/21/98
9/21/98
9/21/98
9/21/98
9/21/98
11/17/98
11/17/98
9/21/98
11/3/98
11/17/98
Date Analyzed TBT DBT MBT
11/3/98 <1 <1 <1
11/3/98 <1 <1 <1
11/3/98 <1 <1 <1
11/3/98 <1 <1 <1
11/3/98 <1 <1 <1
11/3/98 <1 <1 <1
11/17/98 <1 <1 <1
11/17/98 <1 <1 <1
11/17/98 <1 <1 <1
11/17/98 <1 <1 <1
11/17/98 <1 <1 <1
11/17/98 6 1 <1
11/17/98 5 2 <1
11/3/98 <1 <1 <1
11/3/98 <1 <1 <1
11/17/98 <1 <1 <1
Sediment
Sediment samples from sites 2 and 4 in each creek were analyzed for cadmium, cobalt,
chromium, copper, manganese, nickel, lead and zinc. Measured concentrations in most cases
exceeded the sample detection limit (Table 6). In Onancock Creek, The Gulf, and Old Plantation
Creek, the concentrations of all metals were higher at the upstream location (site 4) than the
downstream site. In Hungar Creek, the opposite was true. When compared to concentrations in
sediment from the references sites, the concentrations were generally lower for all metals at the
eastern shore sites than at the reference sites.
23
-------
Table 6. Metals concentrations in sediment samples from sites 2 and 4 in each study creek,
replicate sediment samples from each reference site, with a comparison to Effects
Range Low and Effects Range Median levels of Long et al. (1995). Values exceeding
the ERL are denoted by single underline.
Sample Site
Onancock Site 2
Onancock Site 4
Hungar Site 2
Hungar Site 4
The Gulf Site 2
The Gulf Site 4
Old Plantation Site 2
Old Plantation Site 4
Poropotank, Rep 1
Poropotank, Rep 2
Carter's Creek, Rep 1
Carter's Creek, Rep 2
Carter's Creek, Rep 3
ERL
ERM
Cd
0.01
0.04
0.46
0.09
0.02
0.61
0.01
0.12
0.21
0.18
0.20
0.25
0.23
1.2
9.6
Co
0.13
0.74
6.75
1.58
0.17
6.21
0.17
2.00
5.20
6.55
7.84
8.49
7.65
Cr
<0.69
3.80
19.00
11.00
O.70
38.57
<0.70
5.30
28.55
26.69
44.10
48.20
46.93
81
370
Cu
O.49
1.06
17.39
2.55
<0.50
25.35
0.50
4.04
7.59
7.99
20.03
20.54
19.38
34
270
Mn
12.01
23.79
192.05
44.27
6.48
155.16
2.88
53.29
294.14
262.19
286.16
274.45
225.59
[730]
[1700]
Ni
0.60
1.51
22.15
4.48
<0.30
23.39
<0.30
4.22
11.31
11.58
23.67
25.04
21.30
20.9
51.6
Pb
1.41
3.63
21.15
28.98
1.23
31.13
1.33
7.18
14.92
14.32
31.06
32.53
30.65
46.7
218.0
Zn
2.83
11.19
104.70
20.96
1.83
1 14.94
2.43
28.37
62.72
62.19
126.36
127.80
125.94
150
410
[] values taken from Ingersoll, et al. (1996) which are based on data for the amphipod Hyalella azteca and the midge
Chironomus riparius, both freshwater species rather than marine.
Only nickel exhibited concentrations that equaled the ERL. This occurred in sediment from two
sites: Hungar 2 and The Gulf 4. Similar concentrations of nickel were observed at the Carter's
Creek reference site, but not the Poropotank River reference site. There is virtually no other data
pertaining to Carter's Creek. There is no obvious modem source of nickel at this site, but that
does not preclude an historic source that is unknown.
Comparing the two reference sites, there was approximately twice as much chromium, nickel,
lead, and zinc in sediments from Carter's Creek and three times as much copper. Concentrations
24
-------
of all other metals were virtually identical.
As noted, the concentrations of metals equaled or exceeded the Effects Range Low only for
nickel ( Hungar Creek Site 2 [H2], The Gulf Site 4 [TG4] and Carters Creek [CC]). In no case
was the measured concentration close to the ERM. These stations were also notable for being
among the lowest in % sand and having nearly identical silt-clay concentrations (Table 1).
However, other stations with similar sediment type did not have metals concentrations
approaching the ERL. Regardless, no toxic effects are expected when concentrations are at the
ERL.
All samples contained very low concentrations of organic compounds of interest. Many
analytes were below reported detection limits which ranged from 1-6 ppb (depended on sample
dry weight). Total semi-volatile compounds ranged from 83-807 ppb dry weight (Table 7). When
present, individual polycyclic aromatic hydrocarbons (PAH) were below 60 ppb dry weight.
Similarly, non-polar chlorinated compounds (including PCB) were low in all samples with a
range of
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Table 9 Butyltin concentrations in sediments from two sites in each creek.
Detection Limit 1 ng/g
All concentrations as Cation in ng/g
Matrix Spike 13 ng/g
Sampling Site
Onancock Site 2
Onancock Site 4
Hungar Creek Site 2
Hungar Creek Site 4
The Gulf Site 2
The Gulf Site 4
Old Plantation Site 2
Old Plantation Site 4
Poropotank
Poropotank Replicate
The Gulf 4 Matrix Spike
The Gulf 4 Matrix Spike Duplicate
Blank
Blank
Date Collected
2/9/99
2/9/99
3/16/99
3/16/99
2/8/99
2/8/99
11/17/98
11/17/98
2/3/99
2/3/99
2/8/99
2/8/99
8/4/99
8/10/99
Date Analyzed
8/4/99
8/4/99
8/4/99
8/4/99
8/4/99
8/4/99
8/4/99
8/4/99
8/4/99
8/10/99
8/10/99
8/10/99
8/4/99
8/10/99
TBT DBT MBT
1.6
1.2
11
13
Toxicological characterization
in situ test results
During the field deployment period, there were two relatively small rain events, one on 11 August,
and a second on 17 August (Table 10). The initial rainfall deposited the greatest amount on
Hungar Creek, with 50 mm (~ 2") collected over a two day period, and smaller amounts of rainfall
recorded on the other three creeks. The rainfall on the 17th was smaller and no rain fell at the
sampling site on Hungar Creek from this storm (this does not preclude rain elsewhere within the
watershed but this is unlikely given the small size of the creek). These differences in rainfall
among the locations emphasize the variability in conditions over the Eastern Shore.
30
-------
Table 10. Conditions during the in situ tests with the number of live shrimp and cumulative
number dead. Thirty-six shrimp were deployed on 5 August and monitored for 8 days.
A second group of 36 shrimp was deployed on 15 August and monitored for an
additional 8 days.
Creek
Onancock
Hungar
Date
8/5/98
8/7/98
8/9/08
8/1 1/98
8/13/98
8/15/98
8/17/98
8/19/98
8/21/98
8/23/98
8/5/98
8/7/98
8/9/98
8/1 1/98
8/13/98
8/15/98
8/17/98
8/19/98
8/21/98
8/23/98
Air
Temperature
25.5
29.5
36.0
27.0
29.0
36.0
32.0
22.0
34.0
29.0
22.4
29.0
35.0
31.0
30.0
34.0
33.0
23.0
28.0
360
Water
Temperature
25.6
26.7
28.0
27.4
27.7
28.4
29.0
29.8
28.4
28.8
25.3
26.6
28.4
27.4
28.6
28.6
29.5
28.1
26.0
28.1
Salinity
17
16
16
15
18
17
17
18
18
20
20
20
20
22
21
20
20
22
22
22
Dissolved
Oxygen
8.2
5.5
8.6
10.6
5.9
ND
7.9
10.5
10.4
11.2
5.7
6.3
6.6
5.69
6.36
6.7
5.6
8.2
7.4
8.1
pH
ND
ND
ND
8.1
6.76
8.04
7.26
ND
ND
ND
ND
ND
ND
7.3
7.58
7.57
7.32
ND
ND
ND
Precipitaton
ND
0
0
9.5mm
0
0
26 mm
4 mm
0
0
ND
0
0
50mm
0
0
0
0
0
0
Number of
Live
Shrimp
36
36
35
34
34
36
35
35
35
35
36
35
34
32
32
36
37
37
34
34
Cumulative
Number of
Dead
Shrimp
1
2
3
3
1
1
4
4
1
1
i
1
31
-------
Table 10(cont.).
Conditions during the in situ tests with the number of live shrimp and
cumulative number dead. Thirty-six shrimp were deployed on 5 August and
monitored for 8 days. A second group of 36 shrimp was deployed on 15
August and monitored for an additional 8 days.
Creek
The Gulf
Old
Plantation
Date
8/5/98
8/7/98
8/9/98
8/1 1/98
8/13/98
8/15/98
8/17/98
8/19/98
8/21/98
8/23/98
8/5/98
8/7/98
8/9/98
8/1 1/98
8/13/98
8/15/98
8/17/98
8/19/98
8/21/98
8/23/98
Air
Temperature
22.6
28.0
33.0
34.0
30.0
32.0
33.0
24.0
34.0
36.0
21.8
29.0
30.0
31.0
32.0
32.0
35.0
25.0
24.0
33.0
Water
Temperature
24.0
26.0
28.4
26.7
28.1
28.6
31.4
28.1
25.1
27.7
24.8
26.6
29.3
28.8
29.2
29.4
32.4
287
257
277
Salinity
24
22
21
21
21
22
21
21
22
21
23
22
20
23
23
23
21
23
25
32
Dissolved
Oxygen
5.5
64
7.2
6.6
6.95
ND
7.8
7.5
7.6
ND
4.4
4.8
5.6
5.93
6.3
ND
9.2
9.2
6.6
7.0
PH
ND
ND
ND
7.12
7.7
7.9!
7.66
ND
ND
ND
ND
ND
ND
7.19
7.62
7.78
7.65
ND
ND
ND
Precipitaton
ND
0
0
28
0
0
2
0
0
0
ND
0
2
11
0
0
10
0
0
0
Number of
Live
Shrimp
36
34
34
34
32
36
36
36
36
35
36
37
36
36
36
36
36
36
36
36
Cumulative
Number of
Dead
Shrimp
1
1
1
3
1
Water temperature increased during the study period from 24 to over 30°C, with slight differences
in temperature among the four creeks (Table 10). Salinity was consistent over time within each
creek, but differed from creek to creek. The average salinity was 17.2 psu at the Onancock Creek
site, 20.9 psu at the Hungar Creek site, 21.6 psu at The Gulf site, and 23.5 psu at the Old
Plantation Creek site. Oxygen concentrations were always above 4 mg/1, and at times above
saturation. pH was usually between 7 and 8. These conditions are all well within the tolerance
limits for the test species.
During the initial 8-day exposure, survival was 91.7% in Onancock Creek, 88.9% in Hungar
Creek, 91.7% in The Gulf, and 100% in Old Plantation Creek. The mortalities were somewhat
32
-------
associated with the rain event on 11 August, but the magnitude of the mortalities were small
compared to what has been seen in seaside creeks at other times (Luckenbach et al, 1996). During
the second 8-day exposure period, single crabs died in Hungar Creek and The Gulf, and the deaths
were not associated with a measurable rain event.
Toxicity of Water Samples
The water from The Gulf and Hungar Creeks (September 1998) showed apparent toxicity to
Palaemonetes pugio larvae after 4-5 days of exposure (Table 11). During the remainder of the
8-day test, mortality occurred daily. The onset of mortality may reflect the time of a molt when
decapods are considered particularly vulnerable, but no observations were made to corroborate
this. Survival for the control group in artificial seawater was 92.5% (range 80% - 100%), well
above the test criterion. Survival in water from all sites in both the Gulf and Hungar Creek was
significantly different from that in the control water in a Dunnett's Test. Using a Kruskal-Wallis
analysis, there were no significant differences in responses among the sites, but only The Gulf
station 2 and Hungar stations 4 and 5 were significantly different from the control group. Survival
in the positive control group exposed to the measured LC50 concentration of 0.71 mg Cd/1 was
27.5% after 4 days (data not shown).
33
-------
Table 11 Survival of Palaemonetes pugio larvae exposed to water from each of four Eastern
Shore creeks. Each value represents the mean of four laboratory replicates.
Significant differences from the control are designated with a '*'.
Station
Onancock 1
Onancock 2
Onancock 3
Onancock 4
Onancock 5
Hungar 1
Hungar 2
Hungar 3
Hungar 4
Hungar 5
The Gulf 1
The Gulf 2
The Gulf 3
The Gulf 4
The Gulf 5
Old Plantation 1
Old Plantation 2
Old Plantation 3
Old Plantation 4
Old Plantation 5
Carter's Creek A
Carter's Creek B
Sampling Date
23 Sept 98
21 Sept 98
21 Sept 98
23 Sept 98
21 Sept 98
23 Sept 98
Mean Survival percentages by day
1
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
1 00.0
2
100.0
100.0
100.0
97.5
100.0
100.0
100.0
97.5
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
3
100.0
100.0
100.0
97.5
100.0
97.5
100.0
97.5
97.5
100.0
100.0
97.5
100.0
97.5
97.5
100.0
100.0
100.0
100.0
100.0
100.0
100.0
4
100.0
100.0
100.0
97.5
100.0
95.0
97.5
97.5
87.5
95.0
100.0
82.5
87.5
92.5
95.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
5
100.0
100.0
97.5
97.5
100.0
80.0
92.5
65.0
52.5
70.0
85.0
37.5
67.5
65.0
85.0
100.0
97.5
100.0
100.0
100.0
100.0
100.0
6
100.0
1 00.0
97.5
97.5
100.0
52.5
82.5
47.5
5.0
32.5
65.0
15.0
55.0
25.0
47.5
100.0
97.5
97.5
1 00.0
100.0
97.5
100.0
7
100.0
100.0
95.0
97.5
97.5
32.5
55.0
25.0
0.0
10.0
47.5
12.5
27.5
12.5
30.0
97.5
97.5
97.5
100.0
100.0
97.5
100.0
8
100.0
100.0
95.0
97.5
97.5
32.5
32.5
17.5
0.0
5.0
45.0
10.0
25
12.5
30.0
97.5
97.5
97.5
100.0
100.0
92.5
100.0
In contrast, water from Onancock and Old Plantation creeks did not show apparent toxicity during
the study. Since several treatments did not have any mortality, a valid ANOVA and Dunnett's
Test cannot be performed. However, for all treatments, survival ranged from 95 to 100%. A
Kruskal-Wallis analysis did not indicate any significant differences for water from either creek.
Although we did not determine growth (defined as the difference between final mean weight of
survivors and mean initial weight of a sample from the same experimental population), a
34
-------
comparison of final weights of surviving P. pugio (Table 12) reveals no significant differences
among the various treatments involving sites in all four creeks using Dunnett's test. Since the
larvae at the start of the test were of the same age ± <24 hr and maintained en masse with
consistent feeding, the larvae were of quite consistent, though unmeasured, size at the start. Thus
the final weights in all tests are a reasonable surrogate for growth.
Water samples from the same locations collected a month later (October 1998) did not produce
significant mortality of C. variegatus larvae (Table 13). In Hungar Creek water, survival was
lowest at site 4 (92.5% survival), and statistically different from all other sites and the control in a
Kruskal-Wallis analysis, but this reflects the lack of variance in all other treatments in which
survival was 100%. In the Gulf, survival ranged from 92.5% to 100%, and no statistically
significant differences were detected with the Kruskal-Wallis test. Similarly, water from
Onancock Creek and Old Plantation Creek produced survival rates of 92.5% to 100%.
As with decapod larvae, growth was not determined. Nevertheless, final weights of surviving C.
variegatus (Table 14) were not significantly different from controls at any site. As in the decapod
larval test, fish larvae were intially uniform in size, so final weights are a surrogate for growth.
35
-------
Table 12 Weight / individual for Palaemonetes pugio larvae on day 8 following exposure to
water from each of four Eastern Shore creeks. Each value represents the mean of
four laboratory replicates. Significant differences from the control are designated
with a'*'.
Station
Onancock 1
Onancock 2
Onancock 3
Onancock 4
Onancock 5
Hungar 1
Hungar 2
Hungar 3
Hungar 4
Hungar 5
The Gulf 1
The Gulf 2
The GulfS
The Gulf 4
The Gulf 5
Old Plantation 1
Old Plantation 2
Old Plantation 3
Old Plantation 4
Old Plantation 5
Carter's Creek A
Carter's Creek B
Sampling Date
23 Sept 98
21 Sept 98
21 Sept 98
23 Sept 98
21 Sept 98
23 Sept 98
Mean weight
0.820
0.830
1.250
1.090
0.980
2.408
0.787
1.990
nd
0.600
1.541
0.833
1.629
1.267
0.824
1.090
0.930
0.870
0.990
0.920
1.520
1.080
SD
0.332
0.063
0.450
0.431
0.093
0.742
0.377
0.000
nd
0.100
0.589
0.000
0.260
0.275
0.183
0.335
0.089
0.117
0.298
0.111
0.807
0.250
36
-------
Table 13 Survival of Cyprinodon variegatus larvae exposed to water from each of four
Eastern Shore creeks. Each value represents the mean of four laboratory replicates.
Significant differences from the control are designated with a '*'.
Station
Onancock 1
Onancock 2
Onancock 3
Onancock 4
Onancock 5
Hungar 1
Hungar 2
Hungar 3
Hungar 4
Hungar 5
The Gulf 1
The Gulf 2
The Gulf 3
The Gulf 4
The Gulf 5
Old Plantation 1
Old Plantation 2
Old Plantation 3
Old Plantation 4
Old Plantation 5
Hawaiian Marine Mix A
Hawaiian Marine Mix B
Sampling
Date
14Oct98
12Oct98
12Oct98
14Oct98
12Oct98
14Oct98
Mean Survival percentages by day
I
100.0
100.0
100.0
100.0
100.0
100.0
100.0
97.5
100.0
100.0
100.0
100.0
97.5
100.0
100.0
100.0
100.0
100.0
100.0
95.0
100.0
100.0
2
100.0
100.0
100.0
100.0
100.0
100.0
100.0
97.5
100.0
100.0
100.0
100.0
97.5
100.0
100.0
100.0
100.0
100.0
100.0
92.5
100.0
100.0
3
100.0
100.0
100.0
100.0
100.0
100.0
100.0
95.0
100.0
100.0
100.0
100.0
95.0
100.0
100.0
100.0
100.0
100.0
100.0
92.5
100.0
100.0
4
100.0
100.0
100.0
100.0
100.0
100.0
100.0
95.0
100.0
100.0
100.0
100.0
92.5
97.5
100.0
100.0
300.0
100.0
100.0
92.5
100.0
100.0
5
100.0
100.0
100.0
100.0
100.0
100.0
100.0
92.5
100.0
100.0
100.0
100.0
92.5
97.5
100.0
100.0
100.0
100.0
100.0
92.5
100.0
100.0
6
100.0
100.0
100.0
1 00.0
97.5
100.0
100.0
92.5
100.0
100.0
100.0
100.0
92.5
97.5
100.0
100.0
100.0
100.0
100.0
92.5
100.0
100.0
7
100.0
100.0
100.0
100.0
97.5
100.0
100.0
92.5
100.0
100.0
100.0
100.0
92.5
97.5
100.0
100.0
100.0
1 00.0
100.0
92.5
100.0
97.5
8
100.0
100.0
100.0
1 00.0
97.5
100.0
100.0
92.5
100.0
100.0
100.0
100.0
92.5
97.5
100.0
100.0
100.0
1 00.0
100.0
92.5
100.0
97.5
37
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Table 14 Weight / individual for Cyprinodon variegatus larvae on day 8 following exposure
to water from each of four Eastern Shore creeks. Each value represents the mean of
four laboratory replicates. Significant differences from the control are designated
with a'*'.
Station
Onancock 1
Onancock 2
Onancock 3
Onancock 4
Onancock 5
Hungar 1
Hungar 2
Hungar 3
Hungar 4
Hungar 5
The Gulf 1
The Gulf 2
The Gulf 3
The Gulf 4
The Gulf 5
Old Plantation 1
Old Plantation 2
Old Plantation 3
Old Plantation 4
Old Plantation 5
Hawaiian Marine Mix A
Hawaiian Marine Mix B
Sampling Date
14Oct98
12Oct98
12 Oct 98
14 Oct 98
12 Oct 98
14 Oct 98
Mean Weight
1.03
0.95
1.06
1.22
1.16
1.13
1.06
1.00
1.02
1.09
1.02
1.13
1.12
1.00
0.92
0.10
0.98
1.10
1.15
1.29
1.09
1.170
SD
0.08
0.09
0.07
0.11
0.10
0.20
0.04
0.10
0.25
0.17
0.12
0.11
0.06
0.10
0.14
0.09
0.05
0.16
0.11
0.11
0.033
0.172
38
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Water from the four creeks collected in March of 1999 generally did not adversely affect Mulinia
lateralis embryos. The mean percent normal embryos was 94% or higher in all but two cases
(Onancock Creek site 4 and The Gulf site 4), with a coefficient of variation less than 10% (Table
15). In stark contrast, water from Onancock Creek site 4 and The Gulf site 4 had virtually no
survivors in any replicate, and no normal embryos. Thus, very acute effects were observed, but
there is no information suggesting a possible causation.
Toxicity of Sediment Samples
Sediment from all creeks had no adverse effects on L. plumulosus (Table 16). The 10-day mean
survival at every station was high, averaging 94 to 100% regardless of location. The range of
survival for all replicates for all creeks was 80% to 100%, the same as for the reference sites.
In short tests such as these, with no food added, it is generally accepted that growth cannot be
reliably measured. We did, however, determine final weight, considering that there would likely
be some food material derived from the sediments that in a food limited exposure system, might
result in some differences, albeit the interpretation of such differences would be compromised.
In general, final weights for L. plumulosus were higher than for the reference site (Carter's Creek)
in every test. The lowest mean weight for amphipods exposed to sediment from any creek site was
at least 1.4 times that for the reference site. In part for this reason, after the initial test in this
series, a second reference sediment was used. This sediment was from the Poropotank River at a
site corresponding to that from which AMRL has obtained control sediment in the past. Though
the final weights of amphipods exposed to these sediments was slightly higher than that for those
exposed to Carter's Creek sediment, the differences were not significant in any test.
The highest mean weights were a factor of 2.3 to 2.8 above the lowest mean weights within each
creek. There no significant differences were detected among stations within a creek by Dunnett's
test.
For Cyprinodon variegatus embryo tests, there were no significant adverse impacts on either
percent hatch or percent fry survival (Table 17). Percent hatch for sediment exposed embryos
ranged from 62.5 to 92.5% with a similar range of percent hatch for the reference site sediments.
Percent survival of fry was high, usually above 83%, and always above 77.5%.
While percent hatch was high in nearly all treatments involving creek sediments, reference
sediments resulted in lower than desired percent hatch. In the first test with sediments from Old
Plantation Creek, percent hatch was 62.5%, well below the desired hatch of 80%. A small number
of eggs not used in the test exhibited a similarly poor hatch rate, suggesting that egg quality was
poor. However, since at least two test sediments produced a hatch rate above 80%, one can also
question the quality of reference sediment. Examination of control results with Leptocheirus
plumulosus performed simultaneously does not support a sediment quality concern.
39
-------
Table 15 Percent normal Mulinia later alls embryos when exposed to water from each of the
creek sites. Significant differences from the control are designated with a '*'.
Treatment
Hawaiian Marine Mix
Eastern Shore Reference
Onancock 1
Onancock 2
Onancock 3
Onancock 4
Onancock 5
Hungar I
Hungar 2
Hungar 3
Hungar 4
Hungar 5
The Gulf 1
The Gulf 2
The Gulf 3
The Gulf 4
The Gulf 5
Old Plantation 1
Old Plantation 2
Old Plantation 3
Old Plantation 4
Old Plantation 5
Mean % Normal
99.28
97.57
97.78
98.60
100.00
0.00*
99.24
95.98
99.57
99.42
96.18
95.73
96.74
97.62
94.64
0.00*
94.66
99.31
99.56
99.42
99.58
99.28
Std Dev % Normal
0.75
1.86
2.81
1.35
0.00
0.00
0.69
4.35
0.75
0.50
0.22
2.46
4.59
4.12
4.65
0.00
2.30
0.69
0.76
0.02
0.37
6.22
40
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Table 16. Mean survival and weight / individual for Leptocheirusplumulosus on day 10
following exposure to sediment from each of four Eastern Shore creeks. Each
value represents the mean of four laboratory replicates. Significant differences
from the control are designated with a '*'.
Station
Onancock 1
Onancock 2
Onancock 3
Onancock 4
Onancock 5
Hungar 1
Hungar 2
Hungar 3
Hungar 4
Hungar 5
The Gulf 1
The Gulf 2
The Gulf 3
The Gulf 4
The Gulf 5
Old Plantation 1
Old Plantation 2
Old Plantation 3
Old Plantation 4
Old Plantation 5
Control A (CC)
Control B (CC)
Control B (PR)
Control B' (CC)
Control B' (PR)
Control C (CC)
Control C (PR)
Sampling Date
9 Feb 99
16 Mar 99
8 Feb 99
17Nov98
17Nov98
8 Feb 99
9 Feb 99
16 Mar 99
% Survival
100.0 ± 0.0
99.0 ± 2.2
96.0 ±6.5
I00.0±0.0
100.0 ±0.0
99.0 ± 2.2
97.0 ± 2.7
100.0 ±0.0
96.0 ± 8.9
98.0 ± 2.7
100.0 ±0.0
99.9 ± 2.2
99.0 ±2.2
94.0 ± 5.5
98:0 ± 2.7
94.7 ± 8.7
93.7 ±5.9
96.0 ± 6.0
96.0 ± 6.0
100.0 ±0.0
94.7 ± 5.6
97.0 ±4.5
98.0 ±2.7
95.0 ± 8.7
99.0 ± 2.2
97.0 ±4.0
97.0 ±4.0
Mean Weight
0.27
0.27
0.40
0.55
0.71
0.33
0.42
0.35
0.38
0.74
0.39
0.24
0.52
0.67
0.47
0.29
0.67
0.50
0.55
0.51
0.23
0.17
0.19
0.15
0.21
0.16
0.18
SD
0.01
0.02
0.03
0.02
0.04
0.01
0.03
0.01
0.05
0.04
0.05
0.02
0.01
0.04
0.04
0.15
0.21
0.16
0.24
0.06
0.05
0.02
0.02
0.02
0.01
0.01
0.02
41
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Table 17 Survival data for Cyprinodon variegatus embryos and larvae exposed to sediment from each of
four Eastern Shore creeks. Each value represents the mean of four laboratory replicates.
Station
Onancock 1
Onancock 2
Onancock 3
Onancock 4
Onancock 5
Hungar 1
Hungar 2
Hungar 3
Hungar 4
Hungar 5
The Gulf 1
The Gulf 2
The Gulf 3
The Gulf 4
The Gulf 5
Old Plantation 1
Old Plantation 2
Old Plantation 3
Old Plantation 4
Old Plantation 5
Control A (CO
Control B (CO
Control B (PR)
Control C (CC)
Control C (PR)
Sampling Date
9 Feb 99
16 Mar 99
8 Feb 99
17Nov98
17Nov98
8-9 Feb 99
16 Mar 99
% hatched
80.0
75.0
77.5
70.0
72.5
90.0
92.5
85.0
92.5
82.5
77.5
77.5
80.0
62.5
72.5
85.0
90.0
70.0
67.5
75.0
62.5
75.0
65.0
90.0
80.0
SD
12.25
8.66
4.33
14.14
10.90
7.07
8.29
11.18
8.29
10.90
4.33
14.79
7.07
8.29
10.90
500
10.00
15.81
4.33
1500
829
11.18
8.66
12.25
000
%fry
survival'
100.0
100.0
93.3
100.0
100.0
96.9
86.9
77.5
90.0
93.8
87.1
93.3
94.4
96.4
100.0
1 00.0
100.0
1 00.0
923
92.2
96.4
94.1
83.3
96.4
78 1
SD
0.00
0.00
6.73
0.00
0.00
541
8.83
2278
17.32
10.83
0.77
6.73
9.63
6.19
0.00
0.00
000
0.00
7.78
839
619
592
16.67
6.19
10.36
% live
Fish2
80.0
75.0
72.5
70.0
72.5
87.5
80.0
65.0
82.5
77.5
67.5
72.5
75.0
60.0
72.5
85.0
90.0
70.0
62.5
70.0
60.0
70.0
55.0
87.5
62.5
SD
12.25
8.66
8.29
14.14
10.90
10.90
7.07
18.03
14.79
14.79
4.33
16.39
5.00
7.07
10.90
5.00
10.00
15.81
8.29
17.32
7.07
707
1658
16.39
8.29
% Fr\ Survival = No Frymj/Cumulative No Eggs Hatchedlnj x 100
% Fish Survival = No Fr>,,,j/Initial No Eggs \ 100
42
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In the first test in 1999, fish embryos were examined on receipt, and only eggs judged fertile were
used to set up the test. Again, percent hatch for the reference site sediment was below the desired
80%, and this was also true for a small sample of eggs held in water without sediment (no
replication). As in the previous test, though percent hatch was inadequate, survival of fry was
exceedingly good. In the final test of this series, percent hatch of fry was high, matching the
results for the test creek sediments, and again fry survival was excellent.
Though percent hatch was not acceptable in two exposures, and therefore caution is necessary in
interpreting the test creek responses, there is no evidence of an effect on hatchability or survival of
fry.
The test of sediment pore water with Mulinia lateralis is not a standard procedure, but rather
exploratory. From a strictly operational perspective, the test was relatively easy to perform, the
exposure period was brief, but the processing of samples after exposure was extremely time
consuming.
A reduced percentage of normal embryos was observed among larvae exposed to sediment pore
water from 7 sites, with at least 1 site in each creek (Table 18). The sites producing adverse
effects were Onancock Creek 1 and 2, Hungar Creek 1 and 5, The Gulf 1, and Old Plantation 2
and 3. While survival was reduced at these sites, survival was never less than 9%. The percent
normal observed in pore water from these sites ranged from 11.81 % to 72.12 %. Pore water from
all other creek sites, ranged from 89.9% to 99.3%. In many cases, the coefficient of variation was
larger than in the test with overlying water, and in several cases equaled or exceeded 10%.
The artificial sea water control (Hawaiian Marine Mix) used for the concurrent analyses of
overlying water using the same batch of larvae served as the control in this exposure series as
well. A second control series in conjunction with the reference chemical test had nearly the same
response with 98.5% normal. A pore water sample from outside the creeks was also examined as a
reference material, but since there is no long experience with the testing of pore water, we cannot
assert a priori that this pore water does not produce adverse effects. The percent normal for pore
water from the reference site was 78% with a coefficient of variation of about 14%. This percent
normal was not significantly different from that of the control in any Kruskal-Wallis test
involving a series of treatments.
43
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Table 18 Percent normal Mulinia lateralis embryos when exposed to pore water from sediment
collected at each of the creek sites. Significant differences from the control are
designated with a '*'.
Treatment
Hawaiian Marine Mix
Eastern Shore Reference
Onancock 1
Onancock 2
Onancock 3
Onancock 4
Onancock 5
Hungar 1
Hungar 2
Hungar 3
Hungar 4
Hungar 5
The Gulf 1
The Gulf 2
The Gulf 3
The Gulf 4
The Gulf 5
Old Plantation 1
Old Plantation 2
Old Plantation 3
Old Plantation 4
Old Plantation 5
Mean % Normal
99.28
77.96
0.00*
72.12*
97.27
91.92
92.96
37.09*
93.03
95.35
92.63
48.53*
55.44*
96.17
97.21
91.71
89.91
97.41
11.81*
53.26*
97.97
97.85
Std Dev % Normal
0.75
10.88
0.00
9.93
2.37
1.60
2.13
24.16
2.81
0.02
1.16
10.69
38.92
1.65
0.69
11.06
10.67
1.83
6.74
3.61
1.36
2.64
44
-------
Reference Chemical Tests
Tests were performed with specimens of each species used in the laboratory toxicity tests to
determine the 4-day LC50 for these populations of animals against a reference toxicant (Table
19). In three cases, the acute toxicity test was performed with animals from the same population
used for the ambient toxicity tests of creek water, but prior to them. In the fourth case (Mulinia
lateralis), the acute toxicity test was performed simultaneously with the ambient toxicity tests of
creek water. Within each ambient toxicity test, a replicated treatment with the reference chemical
at the estimated LC50 was included.
For Palaemonetes pugio, a single acute toxicity test was performed with larvae produced from one
or more females in the same population that produced larvae for the tests of creek waters. The
LC50 was 0.71 mg/1 (95% confidence range 0.5-1.0 mg/1). Control survival was 100%.
Table 19 Lethal concentrations for Cadmium tests with each test species.
Species
Palaemonetes pugio
Cyprinodon variegatus
Leptocheirus plumulosus
Mulinia lateralis
Test
1
1
2
1
2
•}
j
1
LC50 (LCL, UCL)
0.71(0.5, 1.0)
3.63 (2.42, 7.65)
4.19' (2.23, 115.69)
0.78(0.56, 1.00)
0.78(0.43, 1.08)
1.15(0.85, 1.60)
0.074 (not determinable)
This experiment, with a dose range of 0.47 to 15 mg/1, when analyzed with all
data, yielded a slope of 0 because of lower than expected mortality at the higher
concentrations. When only the data for the lower 4 exposures were analyzed, the
reported results were obtained, but these are judged unreliable. They are presented
only for completeness.
45
-------
Two tests were performed with Cyprinodon variegatus starting with <24 hr old larvae. In the first
test, the LC50 was 3.63 mg/1 (95% confidence range 2.42-7.65 mg/1). Control survival was 100%
and the response slope was significantly different from 0. However, since the mortality rate at the
highest exposure concentration was lower than expected, the test was repeated and an additional
exposure concentration was included on the upper end. In this test, the responses to the first 5
concentrations were generally similar to responses in the first test, and the response to the added
high concentration not higher than that at lower concentrations. If one calculates an LC50 using
the lowest four concentrations, the LC50 was 4.19 mg/1 (95% confidence range 2.23-115.69
mg/1). The LC50 is withing the 95% fiduciary range of the first test, but the range in this test is
greatly increased. What remains unexplained is the lack of dose-response relationship above the
LC50. One might speculate that this results from a solubility issue, but in the absence of
concentration measurements to confirm the exposures, this cannot be verified.
Three toxicity tests were performed with Leptocheirus plumulosus, in July 1998 and July and
August 1999 (both 4 months or more after the final tests of field collected sediment). The LC50
was 1.15 mg/1 in July 1998 and 0.78 in both tests in July and August 1999. Though similar, these
concentrations were significantly different.
The acute test for Mulinia later alls and cadmium was accomplished simultaneously with the
ambient water and pore water exposures. The LC50 was 0.074 mg/1 based on survivorship.
Survival at 0.1 mg/1 consisted of one abnormal individual. At 0.032 mg/1, the percent abnormal
larvae averaged 11 (6-15%). The percent abnormal at lower concentrations and in the control
ranged from 1.5 to 3.6% (0-5.7%). Clearly, the change in percent abnormal is not great even for
the standard toxicant until the acutely lethal concentration was approached or exceeded.
Confidence limits could not be calculated by the inverse regression method recommended in
ASTM Designation E724 and described in Sokal and Rohlf (1969). This inability to produce
confidence limits was a result of high variance in the data and the low number of degrees of
freedom.
46
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DISCUSSION
The eastern shore of Virginia, as a highly agricultural area with significant access to the Bay for
fishing, has been considered an area with low risk of chemical contamination. In 1996, a year with
extremely heavy rainfall, it became apparent that there was significant risk of pulsed inputs of
selected contaminants, especially pesticides used in tomato culture. In modern tomato culture,
raised beds are covered with plastic sheets that funnel runoff into valleys between rows which
then drain rapidly off the field. Tomato fields differ in management of water leaving fields,
ranging from diversion into wooded areas or green ways, retention ponds, and in rare instances
directly into a tidal creek along the shoreline. This pattern of operation has been observed in South
Carolina and elsewhere, where pulsed inputs and adverse effects have been observed (Scott et al.
1987,1990).
In Virginia, the initial reports of adverse effects now attributed to this agricultural method
occurred in the early 1990's, culminating in 1996 during the period of excessive rainfall. At that
time, a clarn hatchery located on Gargathy Creek about a mile downstream of a tomato field
experienced catastrophic culture failures at all stages of clam development following rain events,
and the owner was able to document direct diversion of field runoff into the creek. While this
creek drains to the ocean, this type of tomato culture is practiced in areas draining into the Bay. A
hatchery located at the mouth of The Gulf also experienced serious culture failure in 1996. A
tomato culture operation is located a short distance upstream, with water from the fields being
captured in a retention pond that in normal years is reported to have minimal release into the creek
(water reuse for irrigation is practiced), but during 1996, there were times when the pond
discharged significant amounts of water to the creek. In response to these events and allegations, a
limited in situ experiment was initiated in an effort to demonstrate the effects of rainfall events on
the grass shrimp, Palaemonetes pugio in the vicinity of tomato culture operations (Luckenbach, et
al., 1996). A major conclusion was that there was significant mortality of shrimp in areas adjacent
to some tomato fields during rain events whereas in areas without tomato fields, mortality was
essential absent. A question that remained was whether there were residues of pesticides
accumulated in the sediments in the creeks that could degrade the quality of the benthic
community.
The Virginian portion of the Bay shore of the Delmarva Peninsula had not previously been the
subject of any efforts to characterize water or sediment. This limited effort was initiated to
provide a preliminary examination of these parameters and to attempt to demonstrate pulsed
impacts through the use of the in situ test.
Chemical characterization of creek water was limited to selected analytes that are relatively
soluble in water and likely to occur in this environment given the major human activities in the
47
-------
area. These included metals and butyltin compounds at two selected sites in all creeks, and
selected chlorinated compounds and butyltin compounds in Hungar Creek and The Gulf in water
exhibiting toxicity in one test.
In no case, were any of the metals found at concentrations exceeding or even approaching water
quality criteria or state water quality standards. The only possible exception to this among the
metals analyzed is mercury for which the detection limit exceeded the chronic water quality
criterion. In all other cases, the detection limit was substantially below the chronic water quality
criteria. Chlorinated hydrocarbons and butyltins were not detected in any water samples from the
two creeks even in samples of water in which toxicity was observed.
Sediment samples from two sites in each creek were also characterized chemically. The metals
thought likely to be present in significant amounts were copper, used extensively in tomato culture
and as an antifoulant in the boating industry, and zinc, also used in agriculture. Yet the only metal
analyte observed to exceed a sediment quality criterion (the ERL), was nickel, found in excess at
one site each in Hungar Creek and The Gulf. The greatest exceedance was only 12%, well short of
the ERM. Similar exceedances were observed in sediment from one of the reference sediment
sites, Carter's Creek.
Sediment samples were also examined for semi-volatile organic compounds chlorinated
hydrocarbons, and butyltins. Low molecular weight semi-volatile compounds were not detected in
any creek. High molecular weight compounds were found above the detection limit, but none in
amounts approaching a sediment quality guideline (when available). Few chlorinated compounds
were detected, and all were pesticides or derivatives of pesticides (hexachlorobenzene, heptachlor,
chlordane, DDT, dieldrin, and endrin) many of which are no longer in use. Concentrations were in
every case low and below sediment quality criteria. The butyltins were not detected despite the
low detection limit (1 ng/g).
These observations are fundamentally consistent with past perceptions that the area is not heavily
contaminated with industrial chemicals. The sediments do not seem to have accumulated
agricultural chemicals to any significant degree despite many years of heavy use of copper in the
potato industry prior to its present use in tomato culture.
From a biological perspective, these systems seem generally "clean" with periodic and isolated
incidences of toxic response. In the in situ study, despite only relatively minor rain events in each
creek, slight mortality coincident in time to these rain events was observed in three of the creeks
(Onancock, Hungar, and The Gulf). These rain events were much smaller in scale than those
during a previous study in the area in which higher mortalities were observed (Luckenbach, et al..
1996). This observation suggests that the concentrations of chemical contaminants were higher
during the 1996 study than the present study, but no water samples were analyzed during a rain
event in either study.
This approach to the study of pulsed events is extremely labor intensive, limiting the number of
sites that can be studied, and the time interval of study. Once one commits to deploying such an
experiment, observation of an effect is a function of the thoughtfulness in site selection and the
48
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probability of a rain event of sufficient magnitude to produce a pulsed input of one or more
contaminants. Without substantial correlated sampling of water during deployment and especially
during rain events, one cannot through this approach demonstrate a correlation between the
biological effect (death) and any of the possible contaminants. A major research need, therefore, is
to develop a simpler approach to field study of pulsed events that can be rapidly and safely
deployed immediately before an event coupled with means of sampling water for chemical
characterization. Some efforts have been made to achieve these ends, especially in the sampling of
water (Scott, et al. 1987, 1990), but these are expensive and not widely used.
Toxicity testing of ambient water and submerged surficial sediments has been used in the Bay
system for over a decade to characterize conditions with respect to toxic materials (Hall, et al.
1991,1992, 1994,1997). Through the use of multiple species with differing sensitivities to
classes of contaminants, one seeks to take a snapshot of ambient conditions. The snapshot of
water conditions relates to a relatively recent time frame, whereas that of sediment may reflect an
integration of contaminant inputs over a longer time scale. Taken together, one obtains a
reasonable impression of ambient conditions. With care in test design, one can exclude effects of
salinity, oxygen, temperature, ammonia, and other less defined parameters unrelated to chemical
contaminants, and therefore it is reasonable to consider these tests to measure toxicity of
contaminants. The tests for water, even with moderately frequent resampling for water
replacement, are unlikely to capture the effect of pulsed inputs, but rather reflect average
conditions.
The endpoints used are easily measured; death, animal size or growth, hatching offish embryos,
and normality of development in bivalve embryos. This list is a mix of common endpoints used
for acute and chronic tests for which there is sufficient familiarity to understand their meaning and
reliability. That is not to say that these are necessarily the most sensitive measures of effect, but
they are cost effective.
In the present study, a toxicological effect was observed only with P. pugio in water collected in
fall of 1998 from all stations in Hungar Creek and The Gulf. There is no evidence of toxic
chemical presence in the suite of analytes examined. All general water quality parameters were
well within normal limits, and ammonia concentration was low. Thus, the observed and rather
acute effects cannot be explained. There are several possibilities for explanation, but no evidence
for or against any of them. For example, there may be one or more chemicals not included in our
analyte list that nevertheless occurred in high concentration. The semi-quantitative relative
retention indices method (Greaves, et al., 1991), though capable of detecting a wide array of
compounds, it still limited by virtue of the extraction procedure and the nature of the detectors
used. Therefore analytes do go undetected. Alternatively, there may have been a disease in the
shrimp larvae. The disease explanation seems unlikely since it would also have resulted in death
among the animals exposed to control water, and that did not occur. Similarly, any type of
laboratory error would most likely have affected the control as well as the experimental animals,
and that did not occur. It would have been desirable to resample and retest these creeks with P.
pugio, but larvae were not available.
There is the possibility that the observation reflects a pulsed input effect, albeit we cannot identify
49
-------
a potential causative agent. To examine this question, an attempt was made to obtain rainfall
information for the period preceding the sampling, but no such records have been found for the
Eastern Shore. The nearest airport with a continuous record is the Norfolk airport, and records
from there are not necessarily good surrogates for rainfall on the eastern shore.
Water taken from the same locations two weeks later and tested with C. variegatus revealed no
similar toxic effects. This difference might reflect a change in ambient water quality or a
difference in sensitivity between the test species. Toxicological effects were not observed with
either species at any site in Onancock and Old Plantation Creeks.
A truly synoptic sampling of all sites was used for the test with the bivalve embryo of M. later alls.
In this test, a high degree of normal development was observed (mean of three laboratory
replicates >94%) was observed at all but two stations, Onancock 4 and The Gulf 4. At these sites,
no embryos survived. No explanation can be offered in either case, but clearly, laboratory error is
highly unlikely since all three replicates exhibited no survival.
Sediment samples tested with the amphipod L plumulosus and the fish C. variegatus exhibited no
toxic effects in survival or percent hatch offish embryos. The amphipod tests met all test
acceptability criteria. The fish embryo tests had hatchability of controls slightly below the
acceptability criterion, but that appears to have been the result of poor batches of eggs obtained
from the supplier. Despite that, there were no sites with lower hatchability than the reference sites;
indeed most sites yielded substantially better hatchability than the reference sites.
This calls into question the quality of sediment from the reference sites, hi the initial test with
fish, only sediment from Carter's Creek, long used in this laboratory as a reference material for L.
plumulosus tests, was included in the test. To evaluate whether the poor reference group
performance was site specific, sediment was obtained from the Poropotank River at or near the
site from which the Applied Marine Research Laboratory at Old Dominion University obtains
control sediment and test animals (Joe Winfield, personal communication). In subsequent tests
with sediment from both reference sites, the two sediments yielded comparable results, with
sediment from the Poropotank River yielding slight lower percent hatch than Carter's Creek. As
noted earlier, both creeks are characterized as chemically clean (for the analytes measured there
are few or no exceedances of sediment quality criteria).
For these sediment tests, a suitable reference site with sandy substrate was not included to control
for effects of grain size. In some sense, the sediments from Site 1 in every creek can be considered
a clean reference site since all are overwashed by Bay water at least twice daily. The animal
responses to these sediments in every case were better than the responses to reference sediments,
and in many cases could not be improved upon.
Acknowledgments
A great deal of credit for what was accomplished goes to the cast of many who participated in
various aspects of this effort. Mary Ann Vogelbein was laboratory manager for the toxicity testing
50
-------
activities, keeping track of all the myriad of details that must be attended to in gathering sampling
containers, cleaning and transport to the field crew, acquisition of test animals, test set up and
daily maintenance, maintenance of data records, and clean up. Mary Ann constantly questioned
procedures, operations, etc. to the betterment of the effort. During the project, technical support
was provided by C. Robertson, L. White, F. Arazayus, K. Staron, and S. Moore. All of these
individuals worked long hours over several week long periods of continuous activity during test
lasting from preparation to cleanup in excess of two weeks. The entire crew is noteworthy for
their commitment and attention to detail.
We are also highly indebted to the field crew, ably managed by Gretchen Arnold of the Eastern
Shore Laboratory. She was responsible for all field sampling and performance of the in situ tests.
She was assisted at one time or another by G. Harmon, P.G. Ross, F. O'Brien, R. Bonniwell, J.
Taylor, A. Sterrett, J. Heisler, and S. Miller with various aspects of her role. They played many
roles, assisting with fabrication of the exposure systems, vessel operation, animal collection,
deployment and maintenance of the in situ experiments, and water and sediment collections, all
integrated into an already complex schedule of research activities.
Without the advice and support of Dr. Michael C. Newman, we could not have performed metals
analyses, least of all with the level of detection achieved here. He personally committed after
hours effort over several weekends and evenings to accomplish the metals extractions, for which I
am deeply grateful. Under his direction, the atomic absorption spectrometry was done by D.
Powell and D. Ownby. These two individuals spent many hours on this task when both had other
major tasks demanding their attention. Their effort, dedication, and commitment were essential
and are gratefully acknowledged.
G. Vadas and J. Greene were responsible for the analysis of organic chemicals. G. Vadas played a
major role in analysis of chromatograms and compilation of the data for the appendices. While
this role is normal for both, we must take this opportunity to recognize the high quality of their
effort and their ever positive contribution.
And to all the unnamed others in various support laboratories who provided analytical support,
and students and staff who provided hands incidentally from time to time, a hearty thank you as
well. And while your names go unrecorded, your willingness to accept samples at late hours and
in other ways to provide critical support despite any inconvenience was essential to us.
51
-------
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54
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APPENDIX A
Table A-l Location references and Hydrographic / meteorological conditions for
sampling dates.
Table A-2 List of Aromatic Retention Indices (ARI)
Table A-3 List of Halogenated Retention Indices (HRI)
Table A-4 List of Polar Retention Indices (POI)
Table A-5 Summary Table for Water Samples with Surrogate Recoveries
Table A-6 Summary Table for Sediment Samples with Surrogate Recoveries
Table A-7 Fortified Water Sample Results with Recoveries
Table A-8 Fortified Sediment Sample Results with Recoveries
Table A-9 Standard Reference Material 194la Results with Recoveries
55
-------
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Table A-2 List of Aromatic Retention Indices (ARJ) with corresponding probable compound
identity.
ARI Probable Compound Id
Naphthalene,d8-
1000 Naphthalene
1035 Benzothiophene
1520 Naphthalene,2-methyl-
1615 Naphthalene,1-methyl-
2000 Biphenyl
2030 Naphthalene.ethyl-
2050 Naphthalene,C2H5-
2065 Naphthalene,2,6-dimethyl-
2100 Naphthalene,C2H5-
2150 Naphthalene,C2H5-
2175 Acenaphthylene
2195 Benzene.hexamethyl-
2255 Acenaphthene,d10-
2265 Acenaphthene
2280 Biphenyl,4-methyl-
2300 Biphenyl.S-methyl-
2325 Naphthalene,C3H7-
2345 Dibenzofuran
2360 Naphthalene,C3H7-
2380 Bibenzyl
2395 Naphthalene,C3H7-
2455 Naphthalene,C3H7-
2482 Naphthalene, 1,6.7-trimethyl-
2518 Fluorene
2565 Biphenyl.methyl-
2620 Dibenzofuran.methyl-
2810 Fluorene.methyl-
2935 Dibenzothiophene
3000 Phenanthrene
3030 Anthracene
3235 Dibenzotniophene.metnyl-
3310 Dibenzothiophene.methyl-
3360 Phenanthrene,3-methyl-
3375 Phenanthrene,2-methyl-
3425 Cyclopenta(def)phenanthrene,4H-
3440 Phenanthrene, methyl-
3457 Phenanthrene, 1-methyl-
3605 Naphthalene.phenyl-
3610 Naphthalene,2-phenyl-
3740 Phenanthrene,C2H5-
3790 Base-neutral,MW=178, C2H5
3860 Fluoranthene
4000 Pyrene
4035 Naphthalene.methyl-.phenyl-
4130 Naphthalene, methyl-, phenyl-
4245 Terphenyl ISTD)
4300 Benzo(a)fluorene
4335 Retene
ARI Probable Compound Id
4366 Benzo(b)fluorene
4440 Base-neutral,methyl-202
4725 Binaphthyl,1,1'-(Sstd)
4777 Benzo(b)naphtho(2,1 -d)thiophene
4800 Benzo{ghi)fluoranthene
4811 Benzo{c)phenanthrene
4910 Benzonaphthothiophene
4971 Benz(a)anthracene
5000 Chrysene
5060 Chrysene.tetramethyloctahydro-
5130 Base-neutral, methy I-228
5239 Phthalic acid,di-(2-ethylhexyl) ester
5310 Base-neutral,methyl-228
5390 Phenanthrene,1-phenyl-
5430 Chrysene.trimethyltetranydro-
5738 Benzo(b)fluoranthene
5762 Benzo(k)fluoranthene
5805 BenzoG)fluoranthene
5909 Benzo(e)pyrene
5925 Benzo(a)pyrene,d12
5946 Benzo(a)pyrene
6000 Perylene
6050 Base-neutral,methyl-252
6568 Quaterphenyl.para-
6820 lndeno(1,2,3-cd)pyrene
6860 Dibenz(a,h)anthracene
7000 Benzo(ghi)perylene
60
-------
Table A-3 List of Halogenated Retention Indices (HRI) with corresponding probable compound
identity.
HRI
870
1000
1405
1460
1620
1643
1770
1913
1973
1999
2009
2038
2048
2086
2116
2128
2141
2151
2181
2189
2227
2240
2261
2300
2315
2338
2345
2358
2380
2414
2417
2424
2438
2444
2464
2495
2499
2519
2534
2546
2562
2575
2578
2578
2598
2608
2644
RRF
0.8
0.22
0.27
1
0.27
0.27
0.45
0.45
0.45
1.03
0.45
1.06
0.89
0.58
1.03
0.58
1.03
0.45
0.58
0.58
0.58
1.03
0.58
0.58
0.58
0.58
0.58
0.883
0.58
0.58
0.68
0.883
0.935
0.58
0.68
0.68
0.68
0.68
0.68
0.68
0.68
0.829
0.883
0.58
0.68
0.68
0.68
Probable Compound Id
Benzene.tetrachloro-
2-Chloronaphthalene
PCB-1
Pentachlorobenzene (ISTD)
PCB-2
PCB-3
PCB-4,10
PCB-7,9
PCB-6
Benzenehexachloride.alpha-
PCB-8,5
Benzene.hexachloro-
Anisole.pentachloro-
PCB-19
Benzenehexachloride.beta-
PCB-30 (Sstd)
Benzenehexachloride.gamma-
PCB-11
PCB-18
PCB-17,15
PCB-24,27
Benzenehexachloride.delta-
PCB-16,32
PCB-34
PCB-29
PCB-26
PCB-25
Chlordane(C)
PCB-28,31
PCB-33,20
PCB-53
Chlordene.alpha-
Heptachlor
PCB-22,51
PCB-45
PCB-46
PCB-69
PCB-52
PCB-49
PCB-47,75,48
PCB-65 (Sstd)
Aldrin
Chlordene.gamma-
PCB-35
PCB-44
PCB-37,42,59
PCB-41,64
HRI
2674
2682
2699
2701
2721
2735
2736
2758
2762
2768
2771
2783
2791
2802
2827
2827
2836
2846
2846
2852
2857
2876
2876
2886
2899
2906
2918
2934
2936
2956
2968
2971
2976
2979
2989
2996
3000
3034
3052
3054
3067
3085
3077
3091
3111
3114
3123
RRF
0.68
0.76
0.68
0.935
0.68
0.9
0.68
0.68
0.68
0.76
0.76
0.76
0.76
0.935
0.974
0.68
0.76
0.63
0.76
0.974
0.76
0.741
0.76
0.974
0.76
0.99
0.76
0.974
0.76
0.76
0.63
0.76
0.79
0.83
0.68
0.76
0.63
0.83
0.83
0.792
0.83
0.741
0.83
0.76
0.83
0.712
0.76
Probable Compound Id
PCB-40
PCB-1 03
PCB-67,100
Chlordane(l)
PCB-63
Heptachlor epoxide
PCB-74
PCB-70
PCB-66
PCB-95
PCB-88
PCB-1 21
PCB-91
Chlordane(S)
Chlordane.trans-
PCB-60,56
PCB-92
DDE.2,4'-
PCB-84
Chlordane(S)
PCB-90,101
Endosulfan I
PCB-99
Chlordane,cis-
PCB-119
Nonachlor.trans-
PCB-83
Chlordane(7)
PCB-97
PCB-87,115
DDE,4,4'-
PCB-85
Dieldrin
PCB-1 36
PCB-77
PCB-1 10
DDD,2,4'-
PCB-82,151
PCB-1 35
Endrin
PCB-1 07
Endosulfan II
PCB-149
PCB-1 18
PCB-134
DDD,4,4'-
PCB-122,131
61
-------
Table A-3 (con't.) List of Halogenated Retention Indices (HRI) with corresponding probable
compound identity.
HRI RRF Probable Compound Id HRI RRF Probable Compound Id
3130 0.7 DDT.2,4'-
3135 0.99 Nonachlor.cis-
3143 0.83 PCB-146
3149 0.792 Endrin aldehyde
3162 0.83 PCB-153,132
3163 0.883 Chlordane(K)
3177 0.76 PCB-105
3207 0.89 PCB-179,141
3229 0.83 PCB-130
3231 0.7913 Endosulfan sulfate
3233 0.89 PCB-176,137
3241 0.7 DDT.4,41-
3255 0.83 PCB-138,158
3283 0.89 PCB-178,129
3302 0.89 PCB-175
3312 0.89 PCB-187
3328 0.89 PCB-183
3346 0.83 PCB-128
3356 0.83 PCB-167
3369 0.89 PCB-185
3389 0.89 PCB-174
3390 0.792 Endrin ketone
3407 0.89 PCB-177
3422 0.83 PCB-171,156
3437 0.5 Dicofol
3435 0.43 Methoxychlor
3444 0.93 PCB-157,201,173
3453 0.93 PCB-204 (Sstd)
3463 0.89 PCB-172
3483 0.89 PCB-180
3492 0.411 Diphenyl ether,2,2',4,4'-
tetrabromo-
3519 0.93 PCB-193
3543 1.1 Mirex
3580 0.93 PCB-170,190
3607 0.93 PCB-199
3625 0.93 PCB-196,203
3676 0.89 PCB-189
3721 0.96 PCB-208,195
3742 0.96 PCB-207
3771 0.26 Permethrin.cis-
3781 0.44 Diphenyl ether,2,2',4,4',6-
pentabromo-
3788 0.93 PCB-194
3797 0.26 Permethrin,trans-
3805 0.93 PCB-205
3859 0.44 Diphenyl ether,2,2',4,4',5-
pentabromo-
3906 0.96 PCB-206
4000 1 PCB-209
5000 0.87 Dibenzodioxin.octachloro-
62
-------
Table A-4 List of Polar Retention Indices (POI) with corresponding probable compound identity.
POI Probable Compound Id
3185 Benzoquinoline
3210 Carbazole,9H-
3460 Carbazole,9H-,methyl-
3495 Carbazole,9H-,methyl-
3900 Carbazole,9H-,C2H5-
3920 Base-neutral,aza-202
3980 Base-neutral,aza-202
5040 Benzocarbazole
5110 Benzanthrone/Benzofluorenone
5175 Benzocarbazole
5215 Benzocarbazole
5680 Base-neutral,aza-252
5755 Base-neutral,aza-252
5920 Benzacridine,C2H5-
5955 Cyclopenta(def)chrysen-4-one
6100 Benzacridine,C2H5-
6100 Benzacridine,C2H5-
6635 Dibenzocarbazole
6750 Dibenzocarbazole
6980 Dibenzocarbazole
7030 Indenylanthracenone
7090 Dibenzocarbazole
7145 Dibenzocarbazole
7175 Dibenzocarbazole
7245 Dibenzocarbazole, methyl-
7310 Dibenzocarbazole, methyl-
7350 Dibenzocarbazole, methyl-
7370 Dibenzocarbazole, methyl-
63
-------
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Table A-7 Fortified Water Sample Results with Recoveries
Analyte Identification
Organochlorine Pesticide
1,2Dibromo-3-chloropropane
Hexachlorocyclopentadiene
Ethridiazole
Chloroneb
Propachlor
Trifluarin
Diallate
Diallate
Hexachlorobenzene
PCB 30 - surrogate
Pentachloronitrobenzene
Chlorothalonil
Alachlor
PCB 65 - surrogate
Metolachlor
Chloropropylate
DCPA
Isodrin
Captan
t-nonachlor
Perthane
Chlorobenzilate
Captafol
Dicofol
PCB 204 - surrogate
Mirex
?-permethrin
?-permethrin
PCB 209 - surrogate
Carbamate pesticide
chloropropham
SWEP
PCB 30 surrogate
Linuron
PCB 65 surrogate
Barban
PCB 204 surrogate
Nitrogen/Phosphorous pesticide
dichlorovos
Propachlor
Chloropropham
Trifluralin
Atrazine
propazin
PCB 30 surrogate
Terbacil
Alachlor
PCB 65 surrogate
Metolachlor
Cyanazme
Chlorpynfos
Stirofos
Butachlor
Norflurazon
PCB 204 surrogate
Fenarimol
Recovery (%)
Deionized Water Water Sample HC
74%
37%
116%
117%
225%
71%
79%
86%
62%
58%
96%
161%
227%
70%
161%
61%
140%
129%
148%
161%
189%
141%
96%
102%
90%
112%
225%
241%
79%
91%
76%
258%
92%
88%
122%
88%
35%
66%
41%
33%
54%
49%
154%
77%
59%
100%
77%
64%
52%
48%
75%
38%
39%
36%
38%
29%
112%
153%
256%
96%
100%
103%
90%
79%
112%
188%
244%
88%
175%
48%
153%
175%
103%
192%
208%
147%
38%
42%
90%
100%
144%
144%
52%
65
-------
Table A-7 (con't) Fortified Water Sample Results with Recoveries
Analyte Identification
Chlorinated Herbicide
3,5 dichlorobenzoic acid
Dichlorprop
2,4.0
pentachlorophenol, silvex, chloramben
PCB 30 surrogate
2,4,5-T
2,4-DB
picloram
PCB 65 surrogate
DCPA
PCB 204 surrogate
Recovery (%)
Oeionized Water Water Sample HC
53%
54%
60%
64%
61%
70%
73%
99%
69%
75%
69%
66
-------
Table A-8 Fortified Sediment Sample Results with Recoveries. Analytes in bold type are added
standards. Sediments are from station ON 2-2. Both samples were fortified with PAH
analytes at 20.0 ug and organochlorine pesticide analytes at 500.0 ng each prior to
extraction.
Analyte
Recovery (%) Analyte
ON 2-2 ON 2-2
Recovery (%)
ON 2-2 ON 2-2
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Carbazole
Fluoranthene
Pyrene
p-terphenyl (ISTD)
binaphthyl, 1,1'(Sstd)
Benz(a)anthracene
Chrysene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(a)pyrene
lndeno(1,2,3-cd)pyrene
Dibenzo(a,h)anthrene
Benzo(ghi)perylene
0%
13%
19%
53%
93%
88%
99%
117%
119%
100%
124%
109%
110%
115%
73%
85%
92%
65%
79%
7%
36%
37%
55%
74%
72%
79%
91%
93%
100%
97%
89%
90%
83%
60%
63%
62%
44%
51%
Pentachlorobenzene (ISTD)
a-BHC
b-BHC
PCB-30 (Sstd)
g-BHC
d-BHC
Heptachlor
PCB 65 {Sstd)
Aldrin
Heptachlor Epoxide
t-chlordane
endosulfan I
c-chlordane
DDE,4,4'-
Dieldrin
Endrin
endosulfan II
DDD,4,4'-
Endrin aldehyde
Endosulfan sulfate
DDT,4,4'-
Endrin ketone
Methoxychlor
PCB-204 (Sstd)
100%
65%
68%
40%
92%
76%
74%
60%
77%
64%
92%
45%
102%
93%
75%
51%
90%
72%
36%
8%
80%
59%
5%
86%
100%
63%
46%
82%
107%
90%
72%
74%
55%
77%
90%
62%
96%
97%
91%
65%
96%
76%
36%
11%
84%
65%
3%
91%
67
-------
Table A-9 Recovery Results for Standard Reference Material 194la. Recovery of SRM 194la
certified (bold type) and non-certified analytes following cleanup and fractionation
Compound Id
Naphthalene
Biphenyl
Acenaphthylene
Acenaphthene
Fluorene
Dibenzothiophene
Phenanthrene
Anthracene
Phenanthrene, 3-methyl-
Phenanthrene,2-methyl-
Cyclopenta(def)phenanthrene,4H-
Phenanthrene, methyl-
Fluoranthene
Pyrene
Binaphthyl,1,1'-(Sstd)
Benzo(c)phenanthrene
Benz(a)anthracene
Chrysene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(j)fiuoranthene
Benzo(e)pyrene
Benzo(a)pyrene
Perylene
lndeno(1,2,3-cd)pyrene
Dibenz(a,h)anthracene
Benzo(ghi)perylene
Recovery (%) Compound Id
4%
7%
22%
52%
29%
87%
56%
46%
80%
56%
130%
46%
71%
71%
99%
219%
82%
134%
83%
108%
26%
70%
53%
54%
54%
86%
44%
PCB8
Hexachlorobenzene
PCB18
PCB28
PCB52
PCB49
PCB44
PCB95
2,4' DDE
PCB 101
PCB99
a-chlordane (cis)
t-nonachlor
PCB 87
4,4' DDE
Dieldrin
PCB 110
PCB 149
PCB 118
4,4' ODD
PCB 153
PCB 105
4,4' DDT
PCB 138, 163,
PCB 187, 182
PCB 183
PCB 128
PCB 156
PCB 180
PCB 170, 190
PCB 194
PCB 206
PCB 209
164
Recovery (%)
43%
43%
122%
43%
70%
41%
66%
132%
375%
77%
135%
57%
117%
49%
118%
35%
105%
83%
68%
73%
55%
91%
114%
99%
65%
121%
190%
267%
111%
80%
72%
52%
73%
-------
APPENDIX B
(Provided as electronic copy only)
Consists of data files for toxicity tests as Quattro Pro spreadsheets and for organic chemistry
chromatograms as Excel spreadsheets.
All toxicity test data is included in a subdirectory of the disc so named. Each file name is
descriptive of content: a two-letter species code, the experiment number, and the type of data
included. Since some tests were accomplished simultaneously, some files refer the reader to
another file that contains the appropriate data. Each experiment has multiple files to include field
collection data, daily mortality data, water quality data during test, and additional measurements
made. All sediment tests include sed in the title.
All organic chemical data is grouped in a series of subdirectories indicating the type of data
included. Each subdirectory includes a series of files, one for each sample processed for that data
type.
69
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